1. Screening, separation and analysis for plant constituents

2. Required Text book:
Wagner, H., Bladt, S. & Zgainski, E. M, Plant Drug Analysis.
Richard J. P. Cannnell, Natural Product Isolation.
Robert M. Silverstein and Francis X. Webster, Spectrometric
Identification of Organic Compounds.

3. Introduction
The use of plant-derived medicinal dates back many centuries although it is still under estimation in modern medicine.
Plants remain the most important source of natural drugs.
More than 30% of prescription drugs are natural products.
More than 60% of anticancer and anti-infective drugs are natural products.

4. Introduction Natural substances: Semisynthetic substances:
The main sources of drugs are as follows:
Natural substances:
From plants, microorganisms, animals,- etc.
(totally obtained from nature).
Semisynthetic substances:
These are drugs that are manufactured by partial synthesis.
Synthetic substances:
These are drugs which are manufactured by total synthesis
(i.e. complete synthetic process or processes).

5. Medicinal plants information sources
Information, however can be obtained from one or more of the following sources:
Herbals
Medical botany
Ethno botany
Herbaria (herbarium)
Field exploration
Phytopharmacological surveys

6. Scheme for Pharmacognositic studies of a crude drug
A systematic study of a crude drug under pharmacognositic scheme involves its description on the following lines.
Official titles, synonyms if any
Biological source and family
Geographical source or habital
History and introduction of crude drug
Cultivation, collection, processing for market and storage
Morphological or macroscopical characters

7. Scheme for Pharmacognositic studies of a crude drug
Microscopic and histological studies
Chemical constituents and qualitative chemical tests
Pharmacological actions, therapeutic and other pharmaceutical use and pharmaceutical preparations or formulations
Commercial varieties, substituents and adulterants
Quality control of crude drugs and phyto-pharmaceuticals derived from the natural sources.

8. Cultivation, collection, processing of crude drugs
The advantages of cultivation may be briefly summarized as follows:
It ensures quality and purity of medicinal plants
It gives a better yield and therapeutic quality
Cultivation ensures regular supply of a crude drug
Cultivation of medicinal and aromatic plants leads to industrialization to a greater extent
Cultivation permits application of modern technological aspects such as mutation and hybridization

9. Methods of cultivation
There are two methods of cultivation
Sexual method
Asexual method
Sexual method
In this method, the plant are raised from the seeds and such plants are known as seedlings

10. Advantages of this method
Seedling are long liver and bear more heavily
Seedlings are comparatively cheaper and easier to raise
Propagation from seed is responsible for the production of seedlings of highly superior merits which may be of great importance to specific producers
It is the only method of choice in case other methods can not be utilized

11. Disadvantages of this method
Generally, seedling trees are not uniform in their growth and yielding capacity as compared to grafted trees
They require more time to bear as compared to grafted plants
Cost of harvesting, spraying of pesticides, etc is more compared to grafted trees

12. Asexual method
In this method, the vegetative part of a plant such as stem or root placed in such an environment that it develops into the new plant
Advantages of this method
The plants are uniform in growth and yielding capacity. In case of fruits trees, uniformity in fruit quality makes harvesting and marketing easy.
Plants start bearing carlier as compared to seedling trees
It encourages the disease-resistant varieties of plant
Inferior or unsuitable varieties can be avoided.

13. Disadvantages of this method
In comparison to seedling trees, these are not vigorous in growth and not long-lived
No new varieties can be envolved in this method.
Factors affecting for cultivation
Altitude, temperature and humidity
Rainfall or irrigation
Soil nature and fertility
Fertilizers
Pests and pest-control

14. Collection of plant materials
Leaves and flowering tops are collected just before the flowering stage (maturity)
Flowers are collected just before pollination or before their full expansion
Barks are collected in spring or early summer when the cambium is active
Fruits are collected depending upon the part of the fruit which is pharmaceutically important. They are collected either ripe or half ripe, but fully grown.

15. Collection of plant materials
Roots are collected in the spring
Rhizomes are collected when they contain the maximun quantity of food reserve material and chemical constituents
The unorganized drugs are collected as soon as they ooze out of the plant.

16. Harvesting of plant materials
Harvesting is an important process in cultivation technology. We should pay attention to the type of the drug to be harvested and the pharmaceutical standards which is needed to achieve. It can be done very efficiently by the skilled workers.
The underground drugs like roots, rhizomes and tubers are harvested by mechanical devices such as diggers or lifters. The tubers and roots are washed thoroughly with water

17. Harvesting of plant materials
Arial parts are harvested by binders
Flowers, seeds and small fruits are harvested by a special device called seed stripper
The seaweeds producing agar are harvested by long handled forks

18. Drying of plant materials
Reasons for drying:
To help in their preservation.
To fix their constituents, by preventing reactions that may occur in presence of water.
To prevent the growth of micro-organisms such as bacteria and fungi.
To facilitate their grinding.
To reduce their size and weight.
Insufficient drying favors spoilage by micro-organisms and makes it possible for enzymatic destruction.

19. Methods of drying
Drying is carried out either by natural or artificial methods.
Natural drying: this is accomplished by natural air in sun or shade.
Artificial drying: this is a rapid method done at well-controlled temperature and is accomplished by: i) Oven dry ii) Vacuum dryer iii) Spray dryer iv) Freeze dryer

20. Stabilization
On long storage, enzymatic reactions will slowly destroy the constituents, because the last traces of water can never be removed.
In order to avoid this degradation, the enzymes should be destroyed before drying, a process usually called stabilization.
The most common method being brief exposure (a few minutes only) of the plant material to ethanol vapor under pressure (0.5 atm).

21. Fermentation
Enzymatic transformation of the original plant constituents is sometimes desirable. e.g. Vanilla pods
The fresh material is placed in thick layers, sometimes covered and often exposed to raised temperatures (30-40 °C) and humidity, so as to accelerate the enzymatic processes.
The fermented product must be dried afterwards to prevent attack by microorganisms, e.g. moulds.

22. Physicochemical factors
Moisture: moisture sometimes affects drugs adversely through activating the enzymes (as in cardiac glycosides).
Heat: rise of temperature up to 45 activates the enzymes causing decomposition of active constituents. Volatile oil containing drugs are also affected by higher temperatures, their content decreases.
Air: oxygen of air oxidizes certain constituents of crude drugs, e.g. linseed and lemon oil, it causes rancidity of fixed oils and resinification of volatile oils.
Light: it affects drugs, especially those having marked colours.e.g. yellow colour of Rhubarb changes to reddish tint, white coloured corollas turn brown.

23. Biological factors
Bacteria: cotton fibres are rendered brittle by bacterial attack which makes the cotton wool objectionable and dusty.
Moulds: the mycelium of delicate hyphae produces an unpleasant mass of clinging particles in powdered drugs.
Insects: they seem to attack all drugs but have preferences to certain drugs as ginger, belladonna, kola, liquorice,.....
Insects which infest vegetable drugs include beetles, mites and moths. They render drugs porous and powdery.

24. Methods for controlling insects
Heat treatment: it is the simplest method and is done by exposing the drug to a temperature of it is effective especially for insect eggs which are not affected by insecticides.
Fumigation: this is done by volatile insecticidal agents in closed areas e.g. CCL4, CS2, CN. Most fumigants do not kill eggs of insects. It is advisable to repeat fumigation at intervals to obtain better results.

25. Garbling
After drying the plant materials, it is gabling to remove sand, dust and foreign materials of the plant which may affect the quality of the drug
Types of machines used for grinding crude drugs:
Hammer mill; a common type for grinding crude drugs.
Knife mill; is useful for production of low-dust powders of leaves, barks and roots for subsequent percolation or maceration.
Tooth mill; is used for production of very fine powders.

26. Packing
The morphological and chemical nature of the drug, its use and the effects of climatic conditions during transportation and storage should taken into consideration while packing the drug.
Drugs containing volatile oils should be packed on well closed containers to avoid the loss of the oil.
Cod liver oil is sensitive to sunlight and should be packed in containers which do not permit sunlight
Some drugs are sensitive to moisture by becoming flexible, microbial growth or decomposition active compounds. These drugs should be packed in containers resistant to moisture.

27. Storage of crude drugs
Preservation of drugs depends on their physical and chemical properties. Proper preservation maintains the quality of drugs. All drugs should be preserved in water-proof, rodent-proof and well closed containers.
Most of the drugs need to be preserved at very low temperature
The protection against insects or mould attack is also important
Radiation due to direct sunlight also causes destruction of active compound

28. Storage of crude drugs
A number of drugs absorb moisture during the storage and become susceptible to microbial growth. The moisture increases the bulk of the drug and causes impairment of it quality. The excessive moisture facilitates enzymatic reactions leading to decomposition of active compounds.
Oxygen is also destroy the drugs. So, the drugs should be preserved in well closed containers and air in containers may be replaced by inert gas like nitrogen.

29. Extracts
Extracts can be defined as preparations of crude drugs which contain all the constituents which are soluble in the solvent used in making the extract.
In dry extracts all solvent has been removed.
Soft extracts and fluid extracts are prepared with mixtures of water and ethanol as solvent.
Tinctures are prepared by extraction of the crude drug with five to ten parts of ethanol of varying concentration, without concentration of the final product.

30. Extracts
Plant constituents are usually contained inside the cells. Therefore, The solvent used for extraction must diffuse into the cell to dissolve the desired compounds whereupon the solution must pass the cell wall in the opposite direction and mix with the surrounding liquid.
An equilibrium is established between the solute inside the cells and the solvent surrounding the fragmented plant tissues.
The speed with which this equilibrium is established depends on:
Temperature 2. pH 3. Particle size 4. The movement of the solvent

31. Phytochemical Screening

32. To carry phytochemical screening the following points must be fulfilled:
Selection of promising plant materials.
Proper collection of selected plants.
Authentication of plant material.
Drying of plant materials.
Grinding of the dried plants.
Garbling of the dried plants
Packing, storage and preservation
Extraction and fractionation of constituents.
Methods of separation and purification.
Methods of identification of isolated compounds
(structure elucidation e.g. UV, IR, MS, H-NMR and C-NMR).

33. Selection of promising plant materials:
Before investing time, effort and money in phytochemical screening it is very important to select a promising plant.
The choice of promising plant depends upon the following:
A plant which have a biological activity.
A plant used in folk medicine.
A plant which show a particular toxicities.

34. Proper collection of selected plants
Drug may be collected from:
Wild plants
Cultivated plants.
Wild plant
Cultivated plant
Disadvantage
Advantage
Scattered in large or unlimited area
Present in limited area.
Difficult to reach
Easy to reach
The collector must be highly skilled botanists
The collector must not be skillful person
Deficiency may occur due to continuous collection
Continuous supply

35. The following precautions should be considered during stage of collection:
The proper time of the day, time of the year and maturity stage of collection is particularly important because the nature and quantity of constituents may vary greatly in somespecies according to the season and time of collection.
The collected plant should be free from any contamination
Collecting plants which are free from diseases (i.e. which are not affected by viral, bacterial, fungal infection).

36. Authentication of plant materials
This may be confirmed by:
Establishing the identity by a taxonomy experts.
Collection of a common species in their expected habitat by a field botanist.
By comparing the collecting plant with a voucher specimen ( herbarium sheet)

37. Extraction and fractionation of constituents
There is no general (universal) method for the extraction of plant materials.
The precise mode of extraction depends on:
The texture of the plant material.
The water content of the plant material.
The type of substances to be extracted or nature of active constituents.

38. Extraction: is the separation of medicinally active portion of plants or animal tissues through the use of
selective solvent and suitable methods extraction.
The principal methods of extraction are:
1- Maceration
2- Percolation
3- Infusion
4- Decoction
5- Digestion
6- Continuous hot extraction technique
(Soxhlet extraction process).
7- Liquid-liquid extraction
8- Solvent-solvent ppt.
9- Distillation

39. Continuous hot extraction technique (Soxhlet extraction process)

40. Choice of solvent As a general empirical rule:
Non polar solvents (petroleum ether and hexane) will dissolve non-polar compounds (fats and waxes).
While polar solvents (methanol, ethanol and water) dissolve polar compound (alkaloid salts and sugars).
The affinity of the solute for the organic phase may be greatly increased by using mixture of solvents instead of single ones (sometimes used mixtures of solvent to increase the solubility).

41. Screening of Drugs After a new substance (expected to be a new drug)
has been developed.
The next step is to subject it to a specified set of procedures in experimental animals.
The aim is to determine whether or not the compound possesses the desired action.

42. Phytopharmacological screening:
Antimicrobial activity
Cinnamomum verum (Eugenol)
Thymus vulgaris (Thymol)
Lavendula officinalis (Linalool)
Antineoplastic activity
Catharanthus roseus (Vincrestine, vinblastin)
Taxus brevifolia (Taxol)
Antimalarial:
Cinchona succirubra (Quinine)
Artemisia annua (Artemisinin)

43. Phytopharmacological screening:
● Hypoglycemic:
Garlic (Allium sativum) (Allicin)
● Cardiotonic
Digitalis purpurea (Digoxin and Digitoxin)
Strophanthus kombe (K-strophanthoside)
● Antiarrythemic
Cinchona succirubra (Quinidine)