Production of DrugsVariability in drug activity

INTRODUCTION:Crude drugs which reach the pharmaceutical manufacturing companies have passed through various stages, which influence the nature and amount of active constituents.Various factors affect the quality of drugs. These factors include:

1. Exogenous Factors:a. Environmental factorsb. Cultivated and Wild plantsc. Collection of drugsd. Dryinge. Storage

2. Endogenous Factors:a. Chemical races or Chemodemesb. Changes in chromosomal number

i. Polyploidyii. Extrachromosomal type

c. Mutationd. Hybridizatione. Transgenic Medicinal Plants

ENVIRONMENTAL CONDITIONS

Plant growth, development and nature of quality & secondary metabolites are affected by environmental conditions. These environmental conditions include:

1. Temperature2. Rainfall3. Day length and quality of light4. altitude

Temperature:• It is the major factor controlling development and metabolism of the plants.• Plants are frequently able to exist in a considerable range of temperature.• Many tropical and subtropical plants will grow in temperate regions in

summer months, but lack frost resistance to withstand winter.• Highest temperatures are observed near equators, but the temperature

falls about 1°C for every 200 meter of elevation.• The annual variation in temp. should also be considered.

– Formation of volatile oils is enhanced at higher temp. but very hot weather may lead to loss of oil.

– Mean optimum temp. for nicotine production in Nicotiana tabacum is 20°C.

– Fixed oils produced at low temp. contain fatty acids with higher content of unsaturated fatty acids than those formed at higher temperatures.

Rainfall:• Quality of plants is affected by:

– Annual rainfall– Its distribution through out the year– Its effect on humidity and Water holding properties of the soil

• Variable results have been observed for the volatile oil production under different rainfall conditions.

• Continuous rainfall may lead to loss of water soluble constituents from leaves and roots by leaching. Ex. :– Decreased content of many Alkaloids, Glycosides and Volatile oils

during heavy rain conditions.– Loss of Sennoside A and B content in Cassia angustifolia during

short draught conditions.

Day length and Radiation characteristics:• In wild, plants are grown where their shade requirements are met and

therefore, under cultivation similar shades must be provided.• Light quality determines the amount of Glycosides or Alkaloids in many

plants.– Belladonna, Stramonium and Cinchona need full sunshine to

produce higher amount of alkaloids.– Long exposure of intense light increases the hyoscine content in

Datura.– An anti-tumor alkaloid, Vinblastin, is increased (due to dimerization

of indole alkaloid catharanthine and vindoline) in Catharanthus roseus.

• Under long day conditions, peppermint leaves contain menthone, menthol and traces of menthofuran. Under short day condition, menthofuran is the major constituent of volatile oil.

• Day – Night also changes relative concentrations of volatile oil content.• Type of radiation:

– UV radiation increases phenylpropanoid and volatile content in Ocimum.

– Flavonoids and Anthocyanins are influenced by UV radiations.

Altitude:• Coconut palm needs a maritime climate• Sugarcane is a lowland plant.• Tea, Coffee, Cocoa, Rhubarb, Tragacanth and Cinchona requires

elevation.• Cinchona grown at low level produces no alkaloids.• Bitter principles of Gentian increases with altitude whereas, the alkaloids

of Aconitum napellus and oil content in Thyme & Peppermint decreases.• Pyrethrine content of Pyrethrum increases at higher altitude.

Cultivated and Wild Plants

• Some drugs are obtained exclusively from cultivated plants. Ex.:– Cardamom, Hemp, Ginger, Peppermint, Cinnamon, Fennel,

Cinchona, Opium etc.• In other cases both cultivated and wild plants are used.• Cultivation is essential in case of drugs like Hemp & Opium and some

drugs where quality can not be compromised.• Quality of drugs can be improved by cultivating drugs under controlled

conditions. The improvement may be due to:– Selection of hybrids, species and varieties which have desired

phytochemical characteristics.– Better development of plants due to improved conditions of soil and

control of pests and diseases.– Better treatment facilities after collection.– Better drying conditions after collection.

Soil:Different plant species vary in their soil and nutrients requirement. Three important characteristics of soil are:

– Physical– Chemical and– Microbiological properties.

• Physical factors include:– Variation in particle size– Water holding capacity of soil– pH

• Chemical Factors include:– Effect of nitrogen containing nutrients:

• Nitrogen fertilizers increase the size and Alkaloid content of plants.

• Nitrogen fertilizers increase silymarin content in fruits of Silybum marianum.

• Microbiological Factors:– Microbiology of soil affects the secondary metabolite production.– Agrobacterium helps in the production of hairy roots.

Propagation from Seeds:• Seeds must be collected when perfectly ripe.• If not planted immediately, they should be stored in cool and dry place.

Usually seeds lose their power of germination if dried and stored for a long period of time.– If seeds of Colchicum autumnale are air dried for a few days, only

5% seeds germinate in one year and some may not germinate even for 5 years.

– Erythroxylum coca and E. novogranatence seeds stored at 4°C for 24 days gave respectively 29% & 0% germination.

• Seeds if slow germinating should be soaked in water or 0.2% gibberlic acid for 48 hrs before sowing.

• Sometimes seeds are treated with sulphuric acid for partial removal of tests from the seeds.

Propagation by Vegetative means:Vegetative propagation methods include:• By bulbs (e.g. squill), Corms (e.g. colchicum), Tubers (e.g. aconite) or

Rhizomes (e.g. ginger).• By division:

– Separation of plants which have number of aerial stems or buds into separate parts each having roots and a growing point. Ex: Althea, Rhubarb, gentian, Malefern etc.

• By stolons (e.g. Liquorice)• By cuttings (e.g. mints, coca)• By grafting and budding

– Cinchona ledgeriana is grafted on C. succirubra to produce increased amount of alkaloids.

• By fermentation:– This process is applied for the production of moulds and bacteria

and is extensively used for the production of antibiotics, lysergic acid derivatives and some vitamins.

• By inoculation (e.g. Ergot)• By cell culture followed by differentiation.

Collection

The drugs may be collected from wild or cultivated plants and the task may be undertaken by unskilled native labours (e.g. Epicacuanha) or by skilled workers in a highly skilled manner (e.g. Digitalis, Belladonna and Cinchona). The factors that affect the quality of drugs include:

Season at which drugs are collected:

o The amount and the nature of active constituent are not constant throughout the year.

o Rhubarb contains no anthraquinone derivatives in winter but contain anthranols which on arrival of summer, converted by oxidation into anthraquinones.

o The content of C-glycosides, O-glycosides and free anthraquinones in developing shoots of Rhamnus purshiana varies throughout the year.

The age of plant at the time of collection:

o Camphor accumulates in heartwood as the tree ages. Camphor is ready for collection at the age of 40 year.

o Steroidal sapogenins isolated from young, matured, old and flowering plants of Agave species had successively fewer hydroxyl groups.

o The Glycoside content in Digitalis purpurea leaves varies with age. Purpurea glycoside A is formed last but reaches a constant maximum of 50% of the total glycosides.

o Highest level of total glycosides in D.purpurea is observed in the first year leaves. But glycosides most important medicinally (e.g. lanatoside C) attain their highest level in second year.

o In Mentha piperitta , high proportions of pulegone is reported in young leaves. Menthone and Menthol is reported in mature leaves.

o In Caraway, carvone is present in young plants but dihydrocarvone in older plants.

o In Eucalyptus, monoterpene 1,8-cineole is present in higher amounts in leaves during flowering.

o In Cannabis, young seedlings contain cannabichromene, while adult plants contain tetrahydrocannabinol.

o Morphine content in Papaver somniferum is highest 2.5 to 3 weeks after flowering. The secondary alkaloids (codeine, thebaine, narcotine and papaverine) reach their maximum somewhat earlier.

o The Hyoscine : Hyoscyamine ratio falls from 80% in young seedlings to about 30% in mature fruiting plants.

o In Vanilla, the highest vanillin biosynthesis occurs 8 months after flower pollination.

o In Ammi visnaga, unripe fruits contain highest content of khellin and visnagin.

Day and Night time:

o Composition of number of secondary metabolites varies during the day and night time.

o Daily variation of the alkaloids of the Poppy, Hemlok, Solonaceous plants and Ergot have been reported.

Generally leaves are collected as the flowers are beginning to open.Flowers are collected when they are fully expanded.Underground organs are collected as the aerial parts die down.Leaves, fruits and flowers should not be collected when they are covered with dew or rain.Discolored organs or the organs attacked by insects should not be collected.In case of leaves, the percentage of stalk portion should be within limits prescribed by different pharmacopoeias.In case of roots and rhizomes, certain amount of aerial stem is often collected.

Drying

If enzymatic action is to be encouraged, slow drying at a moderate temperature is necessary.

o Vanilla pods, Cocoa seeds and Gentian roots should be dried at a moderate temperature.

If enzymatic action is not desired, drying should take place as soon as possible after collection.

Drugs containing volatile oils are liable to lose their aroma if not dried or if the oil is not distilled from them immediately. Moist drugs are attacked by moulds; therefore, drying apparatus and distillation units should be situated near the growing plants.

Duration of drying time varies from a few hours to many weeks and in case of open air, drying depends on weather condition.

Open air drying is used for clove, colocynth, cardamom and cinnamon.

Drying in artificial heat is more rapid than open air drying and is often necessary in tropical countries, where humidity is very high.

Rapid drying helps flowers and leaves to retain their color and aromatic drugs their aroma.

As a general rule: o Leaves, herbs and flowers may be dried between 20 to 40°C.o Bark and roots should be dried between 30 to 65°C.

Storage

Long storage of drugs is not recommended. Drugs such as Hemp and Sarsaparilla deteriorate even when carefully

stored. It has been reported that the content of Taxol in Taxus baccata leaves and

extract stored at room temperature for one year decreased by 30 - 40% and 70 -80% respectively.

The Alkamides of popular immunostimulant herb Echinacea purpurea decrease rapidly on storage.

Drugs stored in the usual containers like sacks, bales, wooden cases, cardboard boxes and paper bags, reabsorb about 10 to 12% moisture.

Drugs such as Digitalis and Hemp should never be allowed to air dried otherwise they loose a considerable part of their activity.

o They may be stored in sealed containers with a dehydrating agent. Volatile oils should be stored in sealed, well filled containers in a cool,

dark place. Fixed oils, particularly Cod liver oil, should also be stored in sealed, well

filled containers in a cool, dark place. The air in the container is sometimes replaced by an inert gas.

Air dried drugs are always susceptible to the attack of insects and other pests, so they should be examined frequently during storage.

In order to reduce undesirable microbial contamination and to prevent the development of other living organisms, some plant materials may require sterilization before storage.

Endogenous Factors:

It refers to phytochemical variation within the species. These factors include:

1. Chemical races or Chemodemes2. Changes in chromosomal number

a. Polyploidyb. Extrachromosomal type

3. Mutation4. Hybridization5. Transgenic Medicinal Plants

Chemical races or Chemodemes:

These are defined as chemically distinct populations within a species and have similar phenotype but different genotype and as such identical in external appearance but differ in their chemical constituents. Ex.:

Fixed oils: Normal rapeseed oil contains 20-40% of erulic acid, an acid having an

extra long carbon chain (C22) and one double bond. Its presence in oil makes it unsuitable for edible purpose. Now varieties are being grown which contain no erulic acid.

Production of oil from sunflower seeds has been improved by varieties that yield linoleic acid enriched oil.

Groundnuts, the source of Arachis oil exists as various starins with different proportions of fatty acids.

Genetic variation in safflower produces different varieties of plants with different oil constituents. High oleic acid containing varieties are used for human consumption and high linoleic acid varieties are used as industrial coatings and lubricants.

Cyanogenetic glycosides: A well known chemical race in the cyanogenetic glycoside series is

Almond.o There are different varieties of Prunus communis showing different

morphological forms with and without amygdalin content. o Some varieties have similar characters but differ in the presence or

absence of glycosides.

Alkaloids: In different Duboisia species, hyoscine, hyoscyamine and norhyoscyamine

content varies greatly.

Morphine content in different varieties of Opium plant varies. From Claviceps purpurea, a number of races have been isolated

containing different groups of ergot alkaloids.

Anthraquinones: Different races of Rheum palmatum produces varying content of rhein &

chrysophanol and other hereditary strains produce high and low yield of anthraquinones.

Cardiac Glycosides: The proportion of glycosides derived from digitoxin and gitoxin is very

different in Digitalis plants of different origin.

Changes in Chromosome number

Polyploidy:In some organisms the chromosomes can be paired not in groups but in threes, fours or higher numbers. These are polyploids individuals – triploids, tetraploids octaploids etc.Such polyploids can be derived by the multiplication of the chromosomes of a single species (autoploids) or as a result of the multiplication of chromosomes following hybridization between two species (alloploids). Polyploidy can be artificially induced in many plants by suitable treatment with an alkaloid Colchicine.

Among the natural polyploids, Valerian occurs across Europe in a variety of forms (2n, 4n and 8n) with a wide variability of pharmacological action.

4n Digitalis lanata is reported to contain a relatively high proportion of lanatosides A and B compared with the 2n form.

Extrachromosomal types:Sometimes plants occur with one or more chromosomes extra to the somatic number and these are known as Extrachromosomal types. In some cases Datura stramonium is shown to possess 25 chromosomes in the somatic cells (in Datura n=12, therefore, 2n+1 = 25 i.e. one chromosome extra).

Mutation

Mutation is a sudden change in the genetic make up of a cell.Ionizing radiations and chemicals produce mutation and bring about the chromosomal changes in the plants.Ionizing radiations include:

X-rays and gamma-rays.

Different chemical which produce mutation include: Nitrogen mustard, Sodiun azide, Malice hydrazide, Colchicine, Ethyl methane sulphonate, Methyl methane sulphonate etc.

Hybridization

In plant breeding, hybridization forms a possible means of combining in a single variety the desired characters of two or more lines, varieties or species and producing new and desirable characters not found in either parent.Many hybrid species are being used for the production of alkaloids and glycosides. Ex. Datura, Duboisia, Nicotiana, Papaver, Withania, etc.

Transgenic Medicinal Plants

Nature itself does effect genetic transfer between very dissimilar organisms.Well known crown gall and hairy root disease are the examples of dissimilar genetic transfer.Agrobacterium rhizogens and A. tumefacience produce hairy root and crown gall disease respectively in plants. Such plants are called Transgenic Medicinal Plants which produce varied amounts of active constituents. Ex.

Belladonna (Atropa belladonna) contain higher amount of Hyoscyamine in transgenic plant infected by A. rhizogens.

Hyoscyamus niger contain less amount of hyoscyamine as compared to Belladonna.