EVALUATION OF CRUDE DRUGS IN PHARMACOGNOSY / WHO & ICH GUIDELINES FOR STANDARDIZATION OF HERBAL DRUGS

 EVALUATION OF CRUDE DRUGS

S.Y B. PHARM SEMESTER IV

UNIT – I

What is evaluation of crude drugs?

EVALUATION OF CRUDE DRUGS: Evaluation of crude drugs means Confirmation of its identity, determination of its quality & purity, and detection of nature of adulteration.

IDENTITY is refer to the exact authentic biological source of the drugs can be established by actual collection of the drug from a plant or animal which has been identified by

•      A qualified, Specialized, and experienced personnel. (Botanist, Herbal specialist)

•      Comparison with the standard/Authenticated sample specimen.

QUALITY is refer to intrinsic value of crude drugs i.e. nature & amount of active medicinal principle.  The active principles may be Alkaloids, Glycosides, Carbohydrates, Tannins, Fixed oil, Volatile oil etc. The high grade quality of the drug can be accomplished by

•      Collection of the drug from correct natural sources either wild or cultivated

•      Collect the crude drug at proper time & in an appropriate manner.

•      Collect the required parts of the plants/

•      Prepration of collected drugs by proper cleaning and drying.

•      Proper preservation of the drug in order to avoid contamination microbes, fungi and insects.

PURITY of the drugs depends upon presence and absence of foreign matter whether organic or inorganic

NEED OF DRUG EVALUATION:

Necessary because of three main reason:-

•      Biochemical variation in the drugs

•      Deterioration due to treatment and storage.

•      Substitution and adulteration due to carelessness, ignorance and fraud.

METHODS OF DRUG EVALUATION:

•         Organoleptic Evaluation

•         Morphological Evaluation

•         Microscopic Evaluation

•         Physical Evaluation

•         Chemical Evaluation

•         Analytical Evaluation

•         Biological Evaluation

ORGANOLEPTIC EVALUATION OF CRUDE DRUGS

It is a technique of qualitative evaluation based on the study of sensory profile of whole drug. This methods refers to a drug evaluation by means of organs of senses like color, odor, taste, size, shape, touch & texture.

Color:- The crude drug was kept in day light & was checked with the standard crude drug. The different parts of crude drugs may acquire a variety of color.

Eg. Material derived from aerial parts of the plants is usually green color. The fruits and seeds having a different color. Barks possess brownish grey to brownish black color etc.

E.g. Starch – White, Cinnamon bark – Brown, Senna - Yellowish green, Clove buds - Dark brown, Kalmegh - Dark green, Ginger, Quill - Pale yellow

Odor and Taste:- Odor and taste of crude material are extremely sensitive criteria based on individual perception.

Odor:- place a small portion of the sample in the palm of the hand or a beaker of suitable size & slowly inhale the air over the material.

•      The crude drug may be odorless or pose an odor which may be Distinct and Indistinct.

•      General terms for describing odor are Aromatic, Balsamic, spicy, fruity, pungent etc.

•      Eg. Aromatic odor of umbeliferus fruit.

Taste:- To achieve the taste keep the material in to the mouth over the tongue. The crude drug may possess a characteristics taste or may be tasteless. The different term used to defined taste are….

E.g. Acidic (sour) - Orange peel, Hibiscus, Saccharine (sweet) - Liquorice, Honey

       Bitter - Mostly alkaloids and Glycosides like Gentian, Picrorrhiza etc.

Distinctive sensation to the tongue:-

Linseed - Mucilaginous & oily (soft feeling)

Ginger - Pungent (Warm biting sensation)

Catechu – Astringent

Ipecac - Nauseous (Tending to excite vomitting).

Touch & Texture:- Touch the materials to determine if it is soft or hard. Texture is best examined by taking a small quantity of material and rubbing it between the thumb and forefinger. It is usually described as smooth, rough etc.

MORPHOLOGICAL EVALUATION OF CRUDE DRUGS:

The study of the form of crude drugs is called morphology and determination of the form crude drugs is called morphography. It can be considered as first step towards identity and degree of purity of crude drugs.

Study of morphology includes visual examination of drugs like study of size and shape of various parts of crude drugs.

Size:- The lenth, width, & Thickness of the crude material are of great importance while evaluating. A graduated ruler in mm is adequate for measurement.

Shape:- The crude drug may be having a different shapes like cylindrical, sub cylindrical, Conical, Ovoid, Disk etc.

Examples:- Conical shape – Aconite, Disc shape - Nux – vomica, Ovoid shape – Jalap, Sub cylindrical – Podophyllum.

A] Leaf and leaflet:-

The study of length, width, apex, margin, venation, the texture of leaf and hairs in upper and lower surface. The feel of the surface describes as soft, hairy, and smooth.

B] Bark:-

Bark have two surfaces an outer and inner. The inner surface is usually lighter in color than outer surface.

 The bark having a three shapes…

1. Flat or curved pieces.
2. Single quill
3. Double quill.

Different shapes of bark:-

1. Flat: - When quite flat and very thick. E.g. Quillaia bark

2. Curved: - When curved and slightly concave on the inner side. E.g. Wild cherry bark.

3. Re-curved:- When the concave side is the outer one. E.g. Pomegranate.

4. Channeled: - When deeply concave on the inner side. E.g. Cassia bark.

5. Single quill: - When deeply concave on the inner side that the edges of the bark nearly or quite overlap. E.g. Cinchona.

6. Double quill: - When both edges are separately in rolled.  E.g. Frangula.

7. Compound quill: - When single and double quills are packed inside one another. E.g. Cinnamon.

MICROSCOPIC EVALUATION:

Microscopic evaluation help in the identification of right variety & search for adulteration. Microscopic evaluation allows more detailed examination & mostly used for qualitative evaluation of organized drugs by their known histological characters in entire as well as in powder form.

 Microscopic evaluation include…..

´  Qualitative microscopy.

´  Linear measurement.

´  Determination of leaf constant.

´  Quantitative microscopy.

QUALITATIVE MICROSCOPY:-

Every plant possess a characteristics tissue structure that can be demonstrated through study of tissue arrangements, cell wall structure, cell content (calcium oxalate crystals, fibers, vessels, parenchyma, trichomes etc).

For the effective result, various reagents and stain can be used to distinguished cellular structure.

Eg. A drop of phloroglucinol and concentrated HCL give red stain with lignin. Mucilage are stained pink with ruthenium red. Starches stained blue with iodine solution. 

LINEAR MEASUREMENT:-

These include identification of crude drugs by measurement;

´  Size of starch grains. (e.g. Diameter of starch grains in cassia bark distinguishes from cinnamon bark)

´  Size of the stomata

´  Diameters of phloem fibers (detection of cassia in cinnamon)

´  Width of the vessel (detection of clove stalks in powdered cloves)

´  Length and width of fibers.

DETERMINATION OF LEAF CONSTANT:-

Various parameters studied here are…..

1. Palisade ratio
2. Vein islet numbers
3. Vein termination numbers
4. Stomata
5. Stomatal number
6. Stomatal index
7.  Trichomes & plant hairs
8. Calcium oxalate crystals.

1.      PALISADE RATIO: - It represents the average no of palisade cells beneath each epidermal cell. (Palisade cells are plant cells located on the leaves, right below the epidermis and cuticle. Palisade cells contain the largest number of chloroplasts per cell, 

Eg. Adhatoda vasica –  5.5 – 6.5     

      Azadiracta indica – 3 - 3.5

2.     VEIN ISLET NUMBERS:- The no of vein-islet per square mm of leaf surface midway between midrib and margin. This value is constant for a given species of a plant and used as a characteristics for the identification of allied species.

Eg: Atropa acuminate 8 - 11, 

      Cannabis sativa-   20 – 30

3.     VEIN TERMINATION NUMBERS:- The no of vein let termination per square mm of leaf surface midway between midrib and margin.(unbroken leaf part isrequired) E.g.  Veinlet termination of Indian senna and Alexandrian senna is quite different for each other.

4.     STOMATA: - A stoma is a minute epidermal opening with following Characteristics 1. A central pore 2. Two kidney shaped similar cells containing chloroplasts known as Guard cells and 3. Varying number of subsidiary (epidermal) cells covering the guard cells. A stoma is made up of a pair of identical guard cells, forming a pore in centre through which gaseous exchange takes place.

TYPES OF STOMATA:- Four types

1. Moss type
2. Gymnospermous type
3. Gramineous type
4. Dicotyledonous:-  There are 4 basic types of stomata among the dicotyledonus, these types are distinguished on the basis of the subsidiary cells surrounding the stomata & their arrangements, The four types are as the following, Paracytic, Diacytic, Anisocytic, Anomocytic, Actinocytic.

Paracytic or Rubiaceous or Parallel cell stomata:-

 In this stomata two guard cells covered by two subsidiary cells. 2 of them have their long axes parallel to the pore, which means that the axes of the subsidiary cells are parallel to the axes of the Pore. E.g. Senna.

Diacytic or Caryophyllaceous or cross – celled stomata:-

 In this stomata two guard cells covered by two subsidiary cells having their long axes perpendicular to the pore. E.g. Peppermint.

Anisocytic or Cruciferous or unequal – celled stomata:-

In this stomata number of guard cells is two but covered by 3 or more subsidiary cells, one of them is distinctly smaller than the others. E.g. Hyoscymus niger.

Anomocytic or Raunculaceous orIrregular celled:-

In this stomata the number of guard cell is two but surrounded by varying number of subsidiary cell. E.g. Digitalis.

Actinocytic or Radiate celled stomata:-

Two guard ells are surrounded by radiating subsidiary cells.

5. STOMATAL NUMBER:-

The average number of stomata present per square millimeter of the epidermis is known as stomatal number.

6. STOMATAL INDEX:-

It is the percentage proportion which the no of stomata form to the total no of epidermal cell. (each stomata being counted as one cell).

Stomatal number varies considerably with the age of the leaf but stomatal index is relatively constant for a given species.

Stomatal index can be calculated by using following equation.

S.I= S/E+S*100   

Where,

SI – Stomatal index

S = No of stomata per unit area

E = No of epidermal cell in the same unit area.

7. TRICHOMES OR PLANT HAIRS:-

These are warty outgrowth of epidermal cells. A trichomes consists of two parts…..

·         Roots which is based in the epidermal lining and

·         Body which is outside the epidermal lining.

Types Of Trichomes:- Trichomes are of three types…

Ø  Covering Trichomes

Ø  Glandular Trichomes

Ø  Hydatodes.

Covering Trichomes: - These are long, slender, in shape and are simple. E.g. Nux vomica.

Glandular Trichomes:- Some hairs have multicellular stalk and head the latter is composed of glandular cells. Stick exudation present on the surface of certain leaves and buds are secreted by colleters. Exception like salt secreting glands as found in tamarix and calcium secreting glands of plumbago.                                      E.g. Vasaka, Digitalis.

Hydatodes:-These are of special kind and their examples are London pride,piper betel. They are developed for water absorption or secretion. Vesiculate hairs in which the surface of leaves and young stem.

8. CALCIUM OXALATE CRYSTALS:-

This are organic crystalline compound by virtue of their specific shapes can be utilized for the identification of herbal drug. Due to this reason they are called as diagnostic characters' of the plant.

TYPES OF CALCIUM OXALATE CRYSTALS:-

1. Cubical (Cube shape):- All sides are equal E.g. Senna, Glycyrrhiza.
2. Rhombic (Diamond shape):- E.g. Kurchi, Mimosa.
3. Tetragonal:- Two axes same one different. E.g. Onion
4. Monoclinic:- All three axes are un – equal. E.g. Oak gall, Veratrum
5. Acicular:- Long, slender, pointed bundles. E.g. Cinnamon, Squill
6. Rosettes clusters:- (aggregation of crystals) similar to expanded rose flower. E.g.  Clove, Arjuna
7. Microsphenoidal (Minute in structure):- Very minute, deltoid or arrow shaped crystals occupying full cells in which they occour. E.g. Henbane.

QUANTITATIVE MICROSCOPY:-

LYCOPODIUM SPORE METHOD:- It is an important analytical technique for powdered crude drugs especially when chemical and other evaluation of crude drugs fail as accurate measures of quality. Lycopodium spores are much characterized in shape and appearance & uniform in size (25 um) on average, 94000 spores present per mg of lycopodium powder.

A powder drug is evaluated by this technique if it contains…

Ø  Well defined particles which may be counted eg. Starch grains or pollen grains.

Ø  Single layered cells or tissues, the area of which may be traced under suitable magnification and actual area calculated.

Ø  The objects of uniform thickness the length of which can be measured under suitable magnification and actual area calculated.

The percentage purity of an authentic drug is calculated using the following equation…..

 Percent purity of drug = N*w*94000*100/S*M*P

Where,

N = No of characteristics structures in 26 fields

            W = weight in mg of lycopodium taken

S = no of lycopodium spores in the same 25 fields

           M = weight in mg of the sample, calculated on basis of sample dried at 105c

            P = Standard value of characteristics structure.

PHYSICAL EVALUATION OF CRUDE DRUGS:

Physical method of evaluation is determined wherever possible. These are rarely constant for crude drugs. But may help in evaluation.

PARAMETERS:-

1. Foreign organic matter              
2. Moisture content
3. Ash value                                     
4. Extractive value
5. Swelling factor                              
6. Viscosity
7. Melting point                                 
8. Solubility
9. Optical rotation                             
10. Refractive index
11. Volatile oil content.                   

1.      FOREIGN ORGANIC MATTER:-  The parts of the organs or organs other than those named in the definition and description of the drugs. These may include insects, moulds, earthy material etc. Medicinal plant material should be entirely free from them which is difficult so pharmacopeias provide the limit for the presence of other parts of the plants. Each crude drugs has its own limits for presence of FOM.

E.g. Garlic and saffron should not contain more than 2 %.

2.     Moisture content:- An excess amount of water in medicinal plants material encourage microbial growth and the presence of fungi and insects cause deterioration. Moisture content of crude drug responsible for decomposition of crude drug due to chemical change or microbial attack. Hence it is necessary to determine and control the moisture content of crude drugs.

Some important physical and chemical methods used to determine the moisture content in drugs are…..

     A. Loss on drying       B. Karl Fischer method (chemical method).

 C. Azeotropic distillation method   D. Spectroscopic method.

A] LOSS ON DRYING:- Loss in the weight of the sample is mainly due to the presence of water but also due to the presence of  small amount of volatile material contribute to the weight loss

LOD is determined by heating a drug at 105⁰c in an oven to a constant weight and calculate the loss of weight.

E.g. Digitalis – Not more than 5%w/w ,  Ergot – NMT 8%w/w.

B] KARL FISCHER METHOD:- This is chemical method extensively used for the determination of very small amount of moisture.

E.g. The crude drugs like Digitalis, Ipecacuhna.

The powdered material is exhausted of water with anhydrous solvents like Dioxan and aliquot is taken for titration. The karl Fischer reagent contain solution So2 and pyridine in dry methanol. Iodine is reduced by So2 in the presence of water causing loss dark brown color of the reagent.

C] AZEOTROPIC DISTILLATION METHOD:- Water forms a heterogeneous azeotrope with the solvents like toluene, benzene and xylene such types of binary mixtures can be distilled up to the azeotropic composition. Dean stark apparatus is used for the determination of moisture content,. Organic solvent forms a azeotropic mixture with water Present in the crude drug. When the drug is heated the organic solvent and water is distilled together which is collected in a graduated tube of the apparatus. Water forms a bottom layer being heavier which can be directly collect after complete distillation.

D]   SPECTROSCOPIC METHOD:-

Water will absorb energy at various wavelengths in electromagnetic Spectrum,  this factor is considered as the quantitative determination of moisture, which is done by IR and UV spectroscopic methods. . This method ideal for substances that contain very less quantity of water.

3. ASH VALUE:- :- The residue remaining after incineration is called ash (Ash is a inorganic salts of carbonates, phosphates, Silicates, of sodium, potassium, calcium and magnesium) 

Ø  Physiological ash:- The ash derived from the plant tissue itself .

Ø  Non Physiological ash:-   The residue of extraneous matter adhering to the plant surface (sand, oil, etc).

Ø  Ash value is used to determine quality & purity of a crude drugs. The maximum temperature used for total ash is not more than 450⁰c.

TYPES OF ASH VALUES:-

A] TOTAL ASH VALUE:- It is used for detecting low grade products, exhausted drugs, & excess of sandy & earthy materials.

Procedure:- Accurately weighed 2 gm of the powdered drug was taken in a tarred silica dish and it was incinerated at a temperature not exceeding 450°C until free from carbon. The sample was cooled and weighed. The percentage of ash was calculated with reference to the air dried drug.

B] ACID INSOLUBLE ASH VALUE:- Used for the determination of earthy matter present on roots, rhizomes, & also on the leaves.

Procedure:- The ash obtained described as total ash was boiled for 5 min. with 25 ml of dilute hydrochloric acid. The insoluble matter was collected on an ash-less filter paper and washed with hot water and ignited to constant weight. The percentage of acid insoluble ash was calculated with reference to the air dried drug.

The majority of crude drugs contain calcium oxalate whose quantity varies frequently. Therefore Total ash of a crude drugs vary within wide limits for specimen of genuine drug. e.g. for Rhubarb, Total ash ranges from 8-40% and in these case determination of acid insoluble content is more preferable.

C] WATER SOLUBLE ASH VALUE:- Used to detect either material exhausted by water or not. E.g. Tea leaves, Ginger rhizomes etc.

Procedure:- To the ash obtained as total ash 25 ml water was added and boiled for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water and ignited in a crucible for 15 minutes at a temperature not exceeding 450оC. The weight of this residue was subtracted from the weight of total ash. The content of water-soluble ash with reference to dried drug was calculated.

D] SULPHATED ASH:- Used for the detection of low grade products. This ash is produced after treating the drug with sulphuric acid to get sulphate salts. Temp- more than 600⁰c.

4. EXTRACTIVE VALUES:- It gives an idea about the nature of the chemical constituents present in a crude drug. Useful for estimation of constituents extracted with the solvent used for extraction. Employed for material for which as yet no suitable chemical or biological assay exists. It can be done by following methods: Cold maceration, hot extraction and ethanol.

A] DETERMINATION OF ALCOHOL SOLUBLE EXTRACTIVE:- 5 g of macerated and air-dried coarse powder of drug was mixed with 100 ml of 95% alcohol in a closed flask and kept for 24 hours, shaking frequently during the first 6 hours and then allowed to stand for 18 hours. Thereafter, it was filtered rapidly taking precautions against loss of the solvent. About 25 ml of the filtrate was evaporated to dryness in a tared flat-bottomed shallow dish, dried at 105 deg C and weighed. The percentage of alcohol-soluble extractive was calculated with reference to the air-dried drug.

B] DETERMINATION OF WATER SOLUBLE EXTRACTIVE:-

      Proceeded as directed for the determination of alcohol soluble extractive, using chloroform water I.P. as a solvent.

C] DETERMINATION OF CHLOROFORM SOLUBLE EXTRACTIVE:-

     Proceeded as directed for the determination of alcohol soluble extractive, using chloroform as solvent.

D] DETERMINATION OF PETROLEUM ETHER SOLUBLE EXTRACTIVE:-

Proceeded as directed for the determination of alcohol soluble extractive, using petroleum ether as a solvent.

5. SWELLING FACTOR:-

Useful in the evaluation of crude drugs containing mucilage.

Useful for the detection of purity of the crude drug determination.

Procedure:-

v  Transfer 1 gm of the seeds to a 25ml of stoppered cylinder.

v  Fill up to the 20ml mark on the cylinder with water.

v  Agitate gently and occasionally during 24 hrs & allowed to stand.

v  Measure the volume occupied by the swollen seeds.

6. VISCOSITY:- Viscosity of a liquid is constant at a given temperature and is an index of its composition. So it can be used as a means of liquid drugs standardization.

Examples:-  Liquid paraffin – Kinematic viscosity not less 64-centistokes at 37.8 deg. Pyroxylin – Kinematic viscosity 1100 – 2450 centistokes.

7. MELTING POINT:- It is one of the parameters to judge the purity of crude drugs containing lipids as constituents. They may of animal or plant origin and contain fixed oil, fats, and waxes. The purity of the following crude drugs can be ascertained by determining their melting points in the range shown against each of them.

Examples:- Wool fat - 34 – 44^oC , Bees wax - 62 – 65.

8. SOLUBILITY:-  The presence of adulterant in a drug could be indicated by solubility studies identify by various solvents

1. Alcohol: 5 gm of powdered material along with 100 ml of alcohol are shaken well occasionally for the first 6 hours and kept undisturbed for 18 hours. The liquefied extract thus obtained was concentrated in an vacuum oven and the percentage was calculated with the weight of the drug powder taken.

2. Water: The procedure adopted for solubility percentage of alcohol is used with chloroform water instead of alcohol to get the water solubility.

Examples:- Pure Asafoetida is soluble in carbon disulphide.

9. OPTICAL ROTATION:- Many volatile oil and other natural products have the ability to rotate the plane of polarized light to right or left side, like wise they are called as dextro or levorotatory.  This detection of optical rotation & its magnitude is an important criterion for the evaluation of certain drugs. It is studied by using polarimeter at 20 deg.

10. REFRACTIVE INDEX:- When a ray passes from a one medium to another of different density, it is bent from original path. Thus, the ratio of velocity of light in vacuum to its velocity in a substance is termed as refractive index of the second medium. Depending upon purity, it’s constant for a liquid and can be consider as one of its standardization. Refractive index of a compound varies with the wave length of the incident light, temperature and pressure.

Examples:-  Ricinus communis(Castor) -  1.475-1.527 (RI)

                          Syzygium aromaticum (Clove) - 1.527-1.535 (RI).

11. VOLATILE OIL CONTENT:- Pharmaceutical significance of aromatic drugs is due to their odorous principal that is volatile oils such crude drugs are standardized on the basis of their volatile contain.  It is determined by steam distillation method by using Clevenger apparatus.

Examples:-

Citrus × limon (lemon peel)     -      2.5

Syzygium aromaticum (Clove) -     15

Foeniculum vulgare (fennel)     -     1.4.

CHEMICAL EVALUATION OF CRUDE DRUGS:

Determination of the active constituent in a drug by chemical tests is referred to as chemical evaluation. Chemical evaluation comprises different chemical test and chemical assays.

CHEMICAL TEST:- Are carried out using various chemical reagents to identify the nature and quantity of chemical constituentsPresent in a crude drugs. It may be

1. Qualitative chemical test
2. Quantitative chemical test.

QUALITATITVE CHEMICAL TEST:- involved identification test for various phytochemical constituents.  These test provide information regarding nature of active principles.

Method for chemical evaluation:- Extract obtained using petroleum ether, chloroform, ethanol and water was prepared using thenrespective solvent. These extracts were tested for the presence of active phytochemicals viz: carbohydrates, proteins, amino acids. fixed oils & fats, alkaloids, glycosides, tannins, phytosterols, triterpenoids, falvonoids,,  saponins, by following standard methods.

A] TEST FOR CARBOHYDRATES:-

1.       Molisch’s test:- To 1 ml of test solution added a few drops of 1 % alpha-napthol and 2-3 ml concentrated sulfuric acid. The reddish violet or purple ring formed at the junction of two liquids.

2.      Barfoed’s test:- 2ml of reagent was added to 2 ml of the test solution, mixed & kept in boiling water bath for 1 min. Red precipitate formed indicates the presence of monosaccharide's.

3.      Seliwanoffs test:- To 3 ml of Seliwanoffs reagent was added to 1 ml of the test sample and heated on a water bath for one minute. The formation of rose red color confirmed carbohydrates.

4.      Fehlings test:- Dissolved 2 mg dry extract in 1 ml of distilled water and added 1ml of Fehling’s (A+B) solution, and heated on a water bath for 10 minutes. The brick red precipitate formed.

B] TEST FOR PROTEINS:-

1.      Biuret test:- To 2 ml of the test solution added 5 drops of 1% copper sulphate solution and 2 ml of 10% NaOH .Mix thoroughly. Formation of purple or violet color confirmed proteins.

C] TEST FOR AMINO ACIDS:-

1.       Millon’s test:- Added 5 drops of millons reagent to 1 ml of test solution and heated on a water bath for 10 min, cooled and added 1% sodium nitrite solution. Appearance of red color confirmed the test.

D] FATS AND FIXED OILS:-

 1. To 5 drops of the sample was added 1 ml of 1% copper sulphate solution and a few drops of 10% sodium hydroxide. The formation of a clear blue solution confirmed the test.

E] TEST FOR ALKALOIDS:-

1.       Dragendorff’s Test: - 2 ml of the filtrate+1 ml of Dragendorff’s reagent. Formation of orange or reddish brown precipitate.

2.      Mayer’s Test:-1 ml of test solution + a drop or two of the Mayer’s reagent. White or a creamy precipitate.

3.      Hager’s Test: - 1 ml of test solution + a drop or two of Hager’s reagent formation of yellow precipitate.

4.      Wagner Test: - Two drops of Wagner’s reagent was added to 1ml of the test solution. The formation of yellow or brown precipitate confirmed the test as positive for alkaloids.

F] TEST FOR GLYCOSIDES:-

1. Liebermann’s Test: - 2ml extract + 2ml CHCl3 + 2ml CH3COOH Violet to Blue to Green coloration

2.  Anthraquinones (Borntrager’s Test): - 3ml extract + 3ml Benzene + 5ml NH3 (10%) Pink, Violet or Red coloration in ammonical layer

3. Coumarins: - 2ml extract + 3ml NaOH (10%) Yellow coloration

4. Saponins (Foam Test): - (a) 5ml extract + 5ml H2O + heat = Froth appears

(b) 5ml extract + Olive oil (few drops) = Emulsion forms.

G] TEST FOR TANNINS:-

1.       Ferric chloride Test:- Added a few drops of 5% ferric chloride solution to 2 ml of the test solution. Formation of blue color indicated the presence of hydrolysable tannins.

2.      Gelatin Test:- Added five drops of 1% gelatin containing 10% sodium chloride to 1 ml of the test solution. Formation of white precipitates confirmed the test. 

H] TEST FOR PHYTOSTEROLS:-

1.       Liebermann-Burchard’s Test:- The extract (2 mg) was dissolved in 2 ml of acetic anhydride, heated to boiling, cooled and then 1 ml of concentrated sulfuric acid was added. A brown ring formation at the junction and the turning of the upper layer to dark green color

I] TEST FOR TRITERPENOIDS:-

1. Salkowaski Test:- Approximately 2 mg of dry extract was shaken with 1 ml of chloroform and a few drops of concentrated sulfuric acid were added. A red brown color formed at the interface.

J] TEST FOR FLAVONOIDS:-

1. Shinoda test:- A few magnesium turnings and 5 drops of concentrated hydrochloric acid was added drop wise to 1 ml of test solution. A pink, scarlet, crimson red or occasionally green to blue color appeared after few minutes.

2. Alkaline reagent test:- Addition of 5 drops of 5% sodium hydroxide to 1 ml of the test solution resulted an increase in the intensity of the yellow color which became colorless on addition of a few drops of 2 M hydrochloric acid which indicated the presence of falvonoids.

3. Lead acetate test: - A few drops of 10% lead acetate added to 1ml of the test solution resulted in the formation of yellow Precipitate confirmed the presence of falvonoids.

K] TEST FOR SAPONINS:-

 1. Foam Test: - 5 ml of the test solution taken in a test tube was shaken well for five minutes. Formation of stable foam confirmed the test.

2. Olive oil test: - Added a few drops of olive oil to 2ml of the test solution and shaken well. The formation of a soluble emulsion confirmed the test.

QUANTITAVE CHEMICAL TEST:- These test give the value or amount of active constituent in the crude drugs. Fixed oil, volatile oil & fats can be determined by this method.  These include…..

1. Acid value: - The no of mg of potassium hydroxide required to neutralize the free acid present in 1 gm sample of fat or oil. Used for resin.

2. Saponification value: -The no mg of potassium hydroxide require to neutralize the fatty acid resulting from complete hydrolysis of 1 gm of the sample of oil or fats. Used for lipids.

3. Ester value:- The no mg of potassium hydroxide require to combined with fatty acid which are present in glyceride form 1 gm sample of fat or oil. Used for volatile oil.

4. Iodine value:- The wt of iodine absorbed by 100 parts by wt of the sample of fat or oil.

CHEMICAL ASSAY:- These test give an approximate value of total phytochemical constituents in a crude drugs. The conventional trimetric estimation as applicable to the estimation of alkaloids from drugs, ester & aldehyde content of volatile oil. Gravimetric methods are used for technique of chemical assay. Eg. The alkaloid drugs  which are routinely analyzed for their total alkaloid content such as Morphine in opium , atropine in belladonna, Strychnine in nux vomica, Reserpine in rauwlfia.etc. Cineole in eucaleptos oil, aldehyde in lemon oil, Vitamins in cod liver oil.

The extraction, isolation and purification of drug are also a part of chemical evaluation.

Extraction methods:- maceration, percolation, Decoction etc.

Purification methods:- Sublimation, distillation, crystallization etc.

ANALYTICAL EVALUATION OF CRUDE DRUGS:

CHROMATOGRAPHIC TECHNIQUES:

A] TLC-Thin layer chromatography

B] HPTLC-High performance thin layer chromatography

C] HPLC-High performance/pressure liquid chromatography

D] GLC-Gas chromatography

E] CC-column chromatography

F] Gel permeation chromatography

G] Affinity chromatography.

SPECTROPHPTOMETRIC METHODS:

A] UV- Ultra violet /visible spectroscopy

B] IR-Infra Red spectroscopy

C] NMR-nuclear magnetic resonance spectroscopy

D] MS-Mass spectroscopy.

A] TLC (THIN LAYER CHROMATOGRAPHY):- TLC are micro analytical technique used for the Analysis and determination of herbal medicines/natural products.  TLC is used as an easier method of initial screening with a semi quantitative evaluation together with other chromatographic Techniques. TLC has become increasingly popular for both qualitative and quantitative evaluation of drugs, Give information about the chief constituents of plant drug, drug fingerprint, & identity & purity of drug. Detection of adulterants & substituent's present in drug.

Thin-layer chromatography is a technique in which a solute under—-goes distribution between two phases, a stationary phase acting Through adsorption and a mobile phase in the form of a liquid.The adsorbent is a relatively thin, uniform layer of dry finely powdered material applied to a glass, plastic or metal sheet or plate.

Glass plates are most commonly used. Separation may also be achieved on the basis of partition or a combination of partition and adsorption, depending on the particular type of support, its preparation and its use with different solvent.

Identification can be effected by observation of spots of identical Rf value and about equal magnitude obtained, respectively, with an unknown and a reference sample chromatographed on the same plate. A visual comparison of the size and intensity of the spots usually serves for semi-quantitative estimation.  Rf values refers to the ration of distance travelled by the solute to the distance moved by the solvent on a thin layer adsorbent.  

        RF      =                        Distance travelled by the compound (solute)

                                                           Distance travelled by the solvent

The Rf value very depends on the purity, nature of substance, composition of solvents and impurities. The spots obtained are visualized either by observing in UV light or by using spray reagents like 1% vanillin - sulphuric acid for steroids & terpenoids dragondorffs reagent for alkaloids etc. The advantages of using TLC to construct the fingerprints of herbal Medicines are its simplicity, versatility, high velocity, specific Sensitivity and simple sample preparation. 

B] HPTLC (HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY):- Advanced form of TLC HPTLC is one of the sophisticated instrumental techniques based on the full capabilities of TLC. It is most flexible, reliable and cost efficient separation technique. The advantage of automation, scanning, full optimization, selective detection principle, minimum sample preparation, hypenation, and so on enable it to be powerful analytical tool for chromatographic  information of complex mixtures of pharmaceuticals, natural products, clinical samples, food stuffs, and so on.

With the help of the CAMAG video store system (CAMAG, Switzerland) and TLCQA-UV methods, it is possible to get useful qualitative and quantitative information from the developed TLC plate.

§  HPTLC plates available in the form of pre coats.

§   Silica gel-Gel with very small particle size used as a stationary phase gives rapid separation with Sensitivity.

§  About 36 cm solvent front migration is sufficient to effect proper Separation.

§  Whatmann-HPTLC plates are produced from 4-5μm layer

§  About 7cm distance achieved in about 4 minutes.

§  Sample preparation:HPTLC needs high concentration sample. small amounts of sample need to apply, sample spot size 1 mm in diameter and sample applied by capillaries. 

C] HPLC (High performance liquid chromatography):- High performance liquid chromatography (HPLC), also known as high pressure liquid chromatography, is essentially a form of column chromatography in which the stationary phase consists of small particle (3-50µm) packing contained in a column with a small bore (2-5mm), one end of which is attached to a source of pressurized liquid eluent (mobile phase). The three forms of high performance liquid chromatography most often used are ion exchange, partition and adsorption. In HPLC mobile phase forced to column under high pressure. Derivatisation in HPLC undertaken to increase sensitivity of detection for a given compound. Colum used in HPLC narrow (1 mm or less) flow rate of mobile phase is (100μl /min)

Uses : HPLC is a popular method for the analysis of herbal medicines because it is easy to learn and use and is not limited by the volatility Or stability of the sample compound. In general, HPLC can be used to analyze almost all the compounds in the herbal medicines.like morhine, emetine, Steroids etc. 

Advantages: most versatile, safest.

D] GC (GAS CHROMATOGRAPHY):-

Gas chromatography (GC), also known as gas liquid chromatography (GLC), is a technique for separation of mixtures into components by a process which depends on the redistribution of the components between a stationary phase or support material in the form of a liquid, solid or combination of both and a gaseous mobile phase. GLC separates volatile components by percolating a gas stream over a stationary phase. Carrier gas used as mobile phase is nitrogen, helium.

Principle: - GLC work on the principle of partition.

It is well-known that many pharmacologically active components in herbal medicines are volatile chemical compounds. Thus, the analysis of volatile compounds by gas chromatography is very important in the analysis of herbal medicines.

Advantages.

1.       The GC of the volatile oil gives a reasonable “fingerprint” which can be used to identify the plant.

2.      The composition and relative concentration of the organic compounds in the volatile oil are characteristic of the particular plant and the presence of impurities in the volatile oil can be readily detected.

3.      The extraction of the volatile oil is relatively straightforward and can be standardized and the components can be readily identified using GC-MS analysis.

E] CC-COLUMN CHROMATOGRAPHY:-  Liquid chromatography in which mobile phase in form of liquid passes over the stationary phase packed in a column. Colum is either a glass, metallic column. the column adsorption chromatography is oldest and still practiced to day for extraction process.

F] GEL PERMEATION CHROMATOGRAPHY:-  It is also known as size-exclusion chromatography. seperation occurs not on the basis of adsorption /partition ,but on the effective size of solutes present in a solution for the separation purpose. Stationary phase used are cross linked polymers which give an open network with large number of pores during flow large size particles can’t enters in to pores hence excluded.  Various types of gels are used sofgel, semi-rigid gels, rigid gels.

Use : separates biomolecules, proteins, poly-peptides etc.

G) AFFINITY CHROMATOGRAPHY:- This technique is mainly used for the separation of proteins, enzymes, antigens, antibodies. The adsorbent used is one of biological substance having a specific affinity for other substance.

These two substances are biologically interacting pairs such adsorbent is attached to a porous stationary phase and placed in a column, when mixture containing the other complement of adsorbent passed through stationary phase.

SPECTROSCOIC TECHNIQUES:

A] U.V (ULTRA VIOLET/VISIBLE) SPECTROSCOPY:-

Ultra violet–visible absorption techniques encompass analytical methods based upon measurement of light absorption by substances in wavelength region from 190 to 900 nm 190-380 nm UV region 380-900 nm visible region.

Applications: To find out whether the system is conjugated (the coloured compounds such as βcarotene, crocetin are in system of extensively conjugated pi-electrons). we can analyze variety of pharmaceutical phytoconstituents like Lobeline-244 nm, Morphine 286 nm, Antharaquinone 505nm.

B] IR-Infra Red spectroscopy:-  12,500-400 cm-1, Mid IR- 4000-400 cm-1 Far IR-400-20cm- o meter can be divided in to single and double beam and Fourier transform spectrophotometer(FTIR)

Applications:- To identify the functional groups that are present in the compound.  Identification of drugs, polymorph, Raw materials, Excipients.

C] NMR-NUCLEA RMAGNETIC RESONANCE SPECTROSCOPY:-

Applications:- It gives idea about structural backbone of compound.

13C-NMR - To identify how many types of carbon atoms are present in the compound.

 1H-NMR - To find out how many types of hydrogen atoms are present in the compound and to find out how the hydrogen atoms are connected.

 NMR is imp tool in elucidation of molecular structure

It is applicable in identification of impurities.

D] MS – Mass spectroscopy:- The electron ionization, subsequent fragmentation of molecules, determination of the mass to charge ratio (m/e). And relative abundances of ions which are produced.

Application:-To determine the molecular structure and molecular weight of the compound and to identify the presence of isotopes patterns for Cl and Br.  It helps in identification of drug constituents.

BIOLOGICAL EVALUATION OF CRUDE DRUGS:

When the plant or extract evaluated by various biological method to determine pharmacological activity, potency and toxicity.

The test performed on living organism like entire animal (cat, dog, guinea pig, monkey, rat, mice, and rabbit), isolated living tissue & organisms, and microorganisms (bacterial, fungal growth).

It is also known as bioassay or biological assay.

In this method, the response produced by the test drug on a living system is compared with that of the stranded preparation.

Such an activity is represented in units as International Units (I.U).Dose is termed as International units IU

•       Digitalis 1IU=76mg of standard

•      Vit-A        1IU=0,344 of standard

•      Vit-D        1IU=0.025 of standard

SIGNIFICANCE:

1. The method is generally used when standardization is not done satisfactory by chemical or physical methods
2. When the quantity of the drug /sample are very less than the drugs are evaluated by biological methods.
3. When the chemical nature of the drug is not known but is has an biological action.
4. Drugs which have different chemical composition but same biological activity.

•      Example: Cardiac glycosides are evaluated by this method on cats, frogs or pigeons.

METHODS OFSTUDIES:

1. Toxic----animals are used
2. Symptomatic-----animals are used
3. Tissue------isolated tissue is used

ü  To estimate potency of drug

ü  To conform therapeutic activity.

1. Evaluation of Hypoglycemic activity:- deficiency of glucose in the bloodstream.

Traditional Diabetic drugs -Momordica charantaka, Fenu greek, Gudmar.

Diabetis is induced in animals by Alloxan & Streptazocin Alloxan cause necrosis of pancreatic islet-B cells which shows 180-250mg/ml fasting blood glucose levels

Streptazocin cause formation of streptomycin they produce cytotoxic nitrourcido glucopyranose which cause diabetes.

•      ANIMALS USED: Rabbits, Rats, Mice (4 to 7 days)

•      Dose:-  rats--80mg/kg ,mice-150mg/kg of streptozocin single oral injection 140-180 mg/kg of alloxan for rabbits at marginal ear vein. for rats and mice intraperitoneally. Insulin levels are noted by tests like RIA. ELISA and glucometer.

2. Evaluation of Anti-inflammatory activity:-

•      Inflammation is caused by mechanical. infections, auto-immune

•      Types of inflammations: Rheumatoid arthritis,  gout, dysmenorrhea.

PRINCIPLE: Anti-inflammatory activity is reduction of local edema induced in rat paw by injecting irritant or inflammatory substance

•      Inflammation is induced by carrageenan and croton oil.

Methods 1:- Carrageenan is a muco-polysaccharide isolated from sea moss which induces inflammation by giving through intraperitoneally saline in a dose of 0.1 ml.

•      animal is treated with herbal extract given orally (antagonist)

•      Volume of paw is measured five times with plethysmo meter.

Methods 2:- Here albino rats or mice are used, edema is produced pinna of ear with croton oil(1 ml/ear)

•      After induce herbal extract is added to the same area

•      Edema is mcasured by using veneircallipers and record the changes

3. Analgesic activity of drug:

Hot Plate Method:

Groups of 10 mice of either sex with an initial weight of 18 to 22 g are used for each dose. The hot plate, which is commercially available, consists of a electrically heated surface. The temperature is controlled for 55° to 56°C. This can be a copper plate or a heated glass surface. The animals are placed on the hot plate and the time until either licking or jumping occurs is recorded by a stop-watch. The latency is recorded before and after 20, 60 and 90 min following oral or subcutaneous administration of the standard or the test compound.

4. Testing of anti-ulcer activity:-

Causes of ulcer:- improper diet, alcohol consumption, stress, drugs (NSAIDS)

Traditional drugs like liquorice atropine,hyoscine and in less extent Gafaranate extracted from cabbage juice shows anti ulcer effect

Chemical used to induce ulcer: Alcohol 1 ml/kg orally

                                                                 Aspirin -200mg/kg orally.

5. EVALUATION OF HEAPATO PROTECTIVITY:-

•      ANIMALS USED: Male and Female Albino rats

•      Heapatotoxicity indused by: Chemicals Industrial pollutants ccl4

•      Drugs: (Paracetamol, Rifampicin)

•      PARAMETERS FOR ESTIMATION

1. PHYSIOLOGICAL-HEXOBARBITAL HYPNOSIS

2. BIO-CHEMICAL SERUM ESTIMATION ENZYMES LIKE.

•      SGOT(serum glutamic oxaloacetic transaminase)

•      SGPT (serum glutamic pyruvic oxaloacetic transaminase)

3.BLOOD CHOLESTEROL, TRIGLYCERIDES LEVELS

4.HISTOPATHOLOGICAL METHODS (liver tissue necrosis) for testing cultured heaptocytes are used for In-vitro studies.