Pharmacognosy - 4. Brief Outline of Chemical Groups
Alkaloids, Glycosides, and Volatile Oils in Pharmacognosy
By Mr. Bhosale, SKCP, Kasbe Digraj
Alkaloids
Introduction:
Alkaloids are a large class of nitrogen-containing secondary metabolites found in a wide variety of natural sources, including plants, microbes, and mammals. Alkaloids often contain one or more rings of carbon atoms, usually with a nitrogen atom in the ring. The position of the nitrogen atom in the carbon ring varies with different alkaloids and with different plant families.
They are highly poisonous but in low dose give therapeutic activities. Even though many alkaloids are poisonous (e.g. strychnine or coniine), some are used in medicine as analgesics (pain relievers) or anaesthetics, particularly morphine and codeine.
General Features:
- Basic in nature.
- Bitter taste.
- Derived from amino acids.
- Notable for their diverse pharmacological activities & potent pharmacological activities.
Occurrence & Distribution:
- Alkaloids are predominantly distributed in higher plants (Pteridophytes, Gymnosperms, Angiosperms), being particularly common in certain families of flowering plants. Alkaloids are compounds primarily found in angiosperms.
- Alkaloids are less commonly found in lower plants (Algae, Bryophytes) and are rarely observed in the animal kingdom.
- More than 10,000 different alkaloids have been identified since the discovery of morphine from the opium poppy (Papaver somniferum) in 1806.
- Notably, all plants in the poppy family (Papaveraceae) are believed to contain alkaloids.
- Opium poppy and the ergot fungus (Claviceps) each contain around 30 different types of alkaloids.
- Alkaloid concentrations vary widely, ranging from a few ppm (e.g., Catharanthus roseus contains 3 g of alkaloids per metric ton of leaves) to more than 15% in the trunk bark of Cinchona ledgeriana. Some plants contain only a single alkaloid (e.g., hyoscyamine in the leaves of belladonna), but more often, they yield a complex mixture dominated by one major constituent.
- One of the primary roles of alkaloids is to protect plants from herbivores and pests. The presence of these compounds can deter animals and insects from feeding on the plants due to their toxic and bitter properties. Some alkaloids can inhibit the growth of nearby plants, a phenomenon known as allelopathy.
Alkaloids occur in different part of plant, which are as follows:
- Roots and rhizomes: e.g. Ipecac, Belladonna, Rauwolfia and Aconite.
- Stem: e.g. Ephedra, Lobelia.
- Bark: e.g. Cinchona, Kurchi.
- Leaves: e.g. Vasaka, Vinca, Hyoscyamus, Tobacco
- Fruits: e.g. Opium, Black pepper.
- Seeds: e.g. Colchicum, Nux-vomica
- All parts of plant: e.g. Datura
- Latex: e.g. Opium
Apart from this alkaloids are also obtained from animal sources like Muscopyridine from a musk of a deer, Castoramine from North America Rodent, Canadian beaver, and from bacteria sources like Pseudomonas aeruginosa.
Extraction of Alkaloids:
1. Traditional Methods
On basic principles of solvent extraction and mechanical processes.
a) Maceration
- Process: Plant material is soaked in a solvent (e.g., ethanol or methanol) for an extended period, allowing alkaloids to diffuse into the solvent.
- Usage: Suitable for thermolabile compounds that degrade under heat.
- Limitations: Slow process, requiring large volumes of solvent and extended extraction times.
b) Percolation
- Process: Plant material is placed in a percolator (a column-like apparatus), and a solvent is passed through it repeatedly to extract alkaloids.
- Advantages: Continuous solvent flow ensures more efficient extraction compared to maceration.
- Limitations: Higher solvent usage and potential for solvent loss through evaporation.
c) Soxhlet Extraction
- Process: Plant material is placed in a thimble inside the Soxhlet apparatus, and a boiling solvent is repeatedly condensed and refluxed through the material.
- Advantages: Efficient for exhaustive extraction of non-thermolabile compounds.
- Limitations: Requires a large quantity of solvent, is time-intensive, and can degrade thermolabile alkaloids due to prolonged exposure to heat.
2. Modern Techniques
These methods are faster, more efficient, and often environmentally friendly.
a) Pressurized Liquid Extraction (PLE)
- Process: Uses high pressure and temperature to extract alkaloids with a solvent. The pressure prevents the solvent from boiling, allowing for efficient extraction at elevated temperatures.
- Advantages: Reduces extraction time and solvent usage. Preserves thermolabile alkaloids due to controlled conditions.
- Applications: Suitable for extracting alkaloids like caffeine and nicotine.
b) Sub-Critical Water Extraction (SWE)
- Process: Uses water at temperatures between 100°C and 374°C and pressures above atmospheric levels. At these conditions, water behaves like a polar organic solvent.
- Advantages: Avoids organic solvents, making it environmentally friendly. Effective for polar and semi-polar alkaloids.
- Applications: Used for extracting hydrophilic alkaloids like berberine.
c) Solid-Phase Microextraction (SPME)
- Process: Uses a solid adsorbent material (like a fiber) to extract alkaloids from liquid, gaseous, or solid samples. The alkaloids are desorbed (release) and analyzed.
- Advantages: Solvent-free technique. High sensitivity for trace alkaloid detection. Efficient for small sample volumes.
- Applications: Commonly used for alkaloids in forensic and pharmaceutical analysis.
Identification Tests for Alkaloids:
Composition: Potassium bismuth iodide solution.
Procedure: To 2.0ml filtrate of plant drug extract, 2.0ml of reagent was mixed.
Result: Formation of reddish brown precipitate indicated the presence of alkaloids.
Composition: Saturated solution of Picric Acid
Procedure: To 2.0ml filtrate of plant drug extract, 2.0ml of reagent was mixed.
Result: Formation of yellow colour indicated the presence of alkaloids.
Composition: Potassium mercuric iodide solution.
Procedure: To 2.0ml filtrate of plant drug extract, 2.0ml of reagent was mixed.
Result: Formation of reddish brown precipitate indicated the presence of alkaloids. (Note: Typically yields a creamy white precipitate)
Composition: Iodine + Potassium iodide solution.
Procedure: To 2.0ml filtrate of plant drug extract, 2.0ml of reagent was mixed.
Result: Formation of reddish brown precipitate indicated the presence of alkaloids.
Therapeutic Activity & Pharmaceutical uses of Alkaloids:
Therapeutic activity of very few, but important alkaloids is described below:
- (i) Ergot alkaloids are well known for their oxytoxic effect i.e. they directly cause contractions of uterine smooth muscles and can control bleeding after delivery and maintain uterine firmness.
- (ii) Nux vomica: It is CNS stimulant and is recommended in cardiac failure. It also stimulates respiratory and cardio-vascular system.
- (iii) Rauwolfia roots are known for their antihypertensive activity and used to lower high blood pressure. It also has tranquilling effect.
- (iv) Vinca: Vincristine arrests mitosis (cell-division) at metaphase and used to treat leukemia (Cancer).
- (v) Ipecacunha: It is emitic in action and in small doses it is expectorant.
- (vi) Opium: Morphine from opium latex is hypnotic sedative and strong analgesic.
- (vii) Belladonna is parasympatholytic in action and used to reduce secretion of saliva, gastric juice and also sweat. It also reduces spasms in case of intestinal gripping caused by strong purgatives.
- (ix) Datura: Hyoscine from datura is parasympatnolytic in action being CNS depressant. Hence, cerebral excitement is treated with datura.
Glycosides
Introduction:
- Herbal glycosides are secondary metabolites, where a sugar part (glycone) is bound to a non-sugar part (aglycone or genin) via a glycosidic bond.
- The nonsugar moiety is called aglycone or genin, whereas sugar part is known as glycone.
- The sugar and nonsugar moiety are linked by the glycosidic linkage.
- The aglycone part is responsible for pharmacological action, whereas sugar part is responsible for solubility, cell permeability, and other pharmacokinetic properties.
- In many instances, glycosides are cleaved to generate active aglycones to produce desired effect, whereas in animals, glycosides facilitate the elimination of poison by binding with sugar moiety of the glycoside molecule.
Glycone + Aglycone = Glycoside- This glycosidic linkage is unstable and is vulnerable to hydrolysis (by enzymes like β-glucosidases or by dilute acids, for example).
Classification:
Glycosides are categorized into different groups based on the kind of glycosidic linkage present:
- O-glycosides: The glycone and aglycone parts in the glycoside are connected by oxygen atom (glycosidic bond is via oxygen); and these are the most predominant form occurring in plants.
- C-glycosides: The glycone and aglycone parts in the glycoside are connected by carbon atom (linkage via a carbon); and these glycosides are resistant to hydrolysis.
- S-glycosides: The glycone and aglycone parts in the glycoside are connected by sulfur atom (linkage via sulfur; aglycone must have –SH group) present in glucosinolates (thioglycosides)
- N-glycosides: The glycone and aglycone parts in the glycoside are connected by nitrogen atom. This kind of linkages is present in (linkage via nitrogen; aglycone must have –NH group) nucleosides.
Occurrence & Distribution:
- Glycosides are widely distributed in the plant kingdom, and they can be found in various plant species across different families and genera.
- Their distribution is not limited to specific regions or climates; instead, glycosides are present in plants all around the world. This wide distribution highlights the significance of glycosides in plants and their diverse roles in plant biology.
- Different types of glycosides serve different functions in plants. For example, some glycosides act as defense mechanisms against herbivores and insects, deterring them from consuming the plant. Others play roles in plant-pollinator interactions, attracting specific pollinators to ensure successful reproduction. Additionally, certain glycosides have medicinal properties and are used in traditional medicine and pharmaceuticals.
In plant, Glycosides occur in different parts which are as follows:
- Root: Senega, Satavari, Ginseng
- Rhizome: Rhuhbarb, Diosgennins, Picrorrhiza
- Wood: Quassia
- Bark: Arjunabark, Quillaia, Wild cherry bark
- Leaf: Senna, Gokhru, Bavchi, Digitalis, Stropanthus
- Fruit: Gokhru, Senna, Vanillca
- Seeds: almond, Mustard, Stropanthus
- Entire herb: Brahmi, Chirata
Examples by Plant Family:
- Asteraceae: sesquiterpene lactone glycosides, used for their anti-inflammatory properties.
- Solanaceae: Contains glycoalkaloids, some of these compounds have pesticidal properties.
- Liliaceae: Contains anthraquinone glycosides found in plants like aloe vera and rhubarb, used for their laxative properties.
- Papaveraceae (Poppy Family): Contains alkaloid glycosides such as morphine and codeine found in opium poppy. These compounds have analgesic and narcotic effects.
- Apocynaceae (Dogbane Family): Contains cardiac glycosides found in plants like foxglove (Digitalis). These glycosides are used in the treatment of heart conditions.
Extraction:
- Preparation of Plant Material: Grind the plant material into a fine powder. This increases the surface area for extraction.
- Extraction:
- Cold Extraction (Maceration/Percolation):
- Maceration: Mix the powdered plant material with the chosen solvent (e.g., ethanol) and let it sit for several hours to a few days. Agitate the mixture occasionally.
- Percolation: Pass the solvent through the plant material in a percolator. Collect the liquid that comes out. This method is faster than maceration.
- Hot Extraction (Soxhlet Extraction): Use a Soxhlet extractor, where the solvent is continuously circulated through the plant material for several hours to extract the glycosides.
- Cold Extraction (Maceration/Percolation):
- Filtration: Filter the extracted solution to remove solid plant particles. Use filter paper and a funnel for this purpose.
- Concentration: Use a rotary evaporator to concentrate the filtered solution. This process removes the solvent and leaves behind a concentrated extract.
- Purification: If needed, further purification techniques like chromatography can be employed to separate specific glycosides from the mixture.
Chemical Tests of Glycosides:
Chemical tests are used to identify the presence of glycosides in a sample. Glycosides are organic molecules that have attached glucose or any mono-oligo saccharide unit. They are usually crystalline or amorphous solids, optically active, and soluble in water and alcohol but insoluble in organic solvents like ether, chloroform, and benzene. There are different types of glycosides, and each type has specific chemical tests that can be used to identify them.
Chemical Tests for Anthraquinone Glycosides
To 1 gm of drug add 5–10 ml of dilute HCl boil on water bath for 10 min and filter. Filtrate was extracted with carbon tetrachloride CCl4 /benzene and add equal amount of ammonia solution to filtrate and shake.
Result: Formation of pink or red colour in ammonical layer due to presence of anthraquinone moiety.
To 1 gm of drug, add 5 ml dilute HCl followed by 5 ml ferric Chloride(FeCl3 ) (5% w/v). Boil for 10 min on water bath, cool and filter, filtrate was extracted with carbon tetrachloride or benzene and add equal volume of ammonia solution.
Result: Formation of pink to red colour due to presence of anthraquinone moiety. This is used C-type of anthraquinone glycosides.
1mg drug + 5ml dil. HCL (Mod. 5ml FeCl3 5%)➡ W. bath boil 10Min ➡ Filtrate + Org. Solvent ➡ Sep. Org. layer + Eql ammonia soln ➡ red/pink(aqs. Layer)
Chemical Tests for Saponin Glycosides
A drop blood on slide was mixed with few drops of aq. Saponin solution.
Result: RBC’s becomes ruptured in presence of saponins.
Aqs. drug + Blood drop on slide ➡ Ruptured RBC’s
To 1 gm of drug add 10–20 ml of water, shake for few minutes.
Result: formation frothing which persists for 60–120s in presence of saponins.
1mg drug + 10 ml of water ➡ shake ➡ froth
Chemical Tests for Steroid and Triterpenoid Glycosides
Alcoholic extract of drug was evaporated to dryness and extracted with CHCl3, add conc. H2SO4 from sidewall of test tube to the CHCl3 extract.
Result: Formation of yellow coloured ring at the junction of two liquid, which turns red after 2 min, indicate the presence of steroid moiety.
Alcoholic extract of drug was evaporated to dryness and extracted with CHCl3, add few drops of acetic anhydride followed by conc. H2SO4 from side wall of test tube to the CHCl3 extract.
Result: Formation of violet to blue coloured ring at the junction of two liquid, indicate the presence of steroid moiety.
Alcoholic extract ➡ Evp to dry ➡ Ext with Org solvent + (Lib buch test - few drops of acetic anhydride) ➡ conc. H2SO4 from side ➡ Salk: Yellow turn Red after 2min ┃ Lib buch test: violet to blue ring at junction
Alcoholic extract of drug was evaporated to dryness and extracted with CHCl3, add saturated solution of Antimony trichloride (SbCl3) in CHCl3 containing 20% acetic anhydride.
Result: Formation of pink colour on heating indicates presence of steroids and triterpenoids.
Alcoholic extract ➡ Evp to dry ➡ Ext with Org solvent + Sat. soln Antimony trichloride (contain 20% acetic anhydride) ➡ Pink on Heating
Triterpenes on addition of saturated solution of trichloro acetic acid forms coloured precipitate.
Drug Soln + sat. soln of trichloro acetic acid ➡ coloured precipitate
It forms yellow colour with unsaturated steroids and triterpenes
Drug Soln + Soln of Tetranitro methane ➡ Yellow
Meta dinitrobenzene solution was added to the alcoholic solution of drug containing alkali, on heating it forms violet colour in presence of keto steroid.
Alcoholic Soln of Drug (Alkaline) + Meta dinitrobenzene solution ➡ heat to forms violet
Chemical Tests for Cardiac Glycosides
To the alcoholic extract of drug equal volume of water and 0.5 ml of strong lead acetate solution was added, shaked and filtered. Filtrate was extracted with equal volume of chloroform. Chloroform extract was evaporated to dryness and residue was dissolved in 3 ml of glacial acetic acid followed by addition of few drops of FeCl3 solution. The resultant solution was transferred to a test tube containing 2 ml of conc. H2SO4.
Result: Reddish brown layer is formed, which turns bluish green after standing due to presence of digitoxose.
Alcoholic extract + Eql vol water ➡ + 0.5ml Strong lead acetate ➡ Shake ➡ Filter (Filtrate + Eql vol CHCl3 ➡ CHCl3 Evaporated ➡ Residue + 3ml Glacial acetic acid + few drop feCl3 transferred to TT containing 2ml H2SO4 ➡ Reddish brown layer turns bluish green
To the alcoholic extract of drug equal volume of water and 0.5 ml of strong lead acetate solution was added, shaked and filtered. Filtrate was extracted with equal volume of chloroform and the chloroform extract was evaporated to dryness. The residue was dissolved in 2 ml of pyridine and sodium nitropruside 2 ml was added followed by addition of NaOH solution to make alkaline.
Result: Formation of pink colour in presence of glycosides or aglycon moiety.
Alcoholic extract + Eql vol water ➡ + 0.5ml Strong lead acetate ➡ Shake ➡ Filter (Filtrate + Eql vol CHCl3 ➡ CHCl3 Evaporated ➡ Residue + 2 ml of pyridine and 2 ml sodium nitropruside 2 ml ➡ NaOH ➡ pink colour
Thick section of leaf of digitalis or the part of drug containing cardiac glycoside, when dipped in sodium picrate solution.
Result: it forms yellow to orange colour in presence of aglycones or glycosides.
Thick leaf section ➡ dipped in sodium picrate solution ➡ Yellow to Orange
To the alcoholic solution of drug few drops of NaOH followed by 2% solution of 3,5-dinitro benzoic acid was added.
Result: Formation of pink colour indicates presence of cardiac glycosides.
Alco. Soln + few drops of NaOH ➡ + 2% solution of 3,5-dinitro benzoic acid ➡ pink colour
Chemical Tests for Coumarin Glycosides
To the concentrated alcoholic extract of drug few drops of alcoholic FeCl3 solution was added.
Result: Formation of deep green colour, which turned yellow on addition of conc. HNO3, indicates presence of coumarins.
Conc. Alco. Extract + alcoholic FeCl3 soln➡ deep green ➡ turned yellow on addition of conc. HNO3
The alcoholic extract of drug was mixed with 1N NaOH solution (one ml each).
Result: Development of blue-green fluorescence indicates presence of coumarins.
Alco. Extract + 1N NaOH ➡ blue-green fluorescence
Chemical Tests for Cynophoric Glycoside
Powdered drug was moistened with water in a conical flask and few drops of conc. Sulphuric acid was added. Filter paper impregnated with sodium picrate solution followed by sodium carbonate solution was trapped on the neck of flask using cork.
Result: Formation of brick red colour due to volatile Hydrogen cyanide(HCN) in presence of cynophoric glycosides takes place.
Water moistened drug in flask+ few drop H2SO4➡ Soaked filter paper sodium picrate soln followed by sodium carbonate soln ➡ trap paper on the neck of flask using cork ➡ brick red colour
Chemical Tests for Flavonoid Glycosides
Filter paper dipped in alcoholic solution of drug was exposed to ammonia vapor.
Result: Formation of yellow spot on filter paper.
filter paper dipped in alco drug soln ➡ exposed to ammonia vapor ➡ yellow spot on paper
The alcoholic solution of flavone or flavonol when treated with metallic magnesium (or Zinc) and hydrochloric acid.
Result: gives an orange, red or violet color
alco drug soln ➡ metallic magnesium (or Zinc) and hydrochloric acid ➡ red or violet
Vanillin HCl was added to the alcoholic solution of drug.
Result: formation of pink colour due to presence of flavonoids.
alco drug soln + Vanillin HCl ➡ pink colour
Therapeutic use:
| Sr. | Glycoside Type | Drug Example | Use |
|---|---|---|---|
| 1. | Anthracene Glycosides | Senna, Aloe, Rhubarb, Quassia | Laxative |
| 2. | Cardiac Glycosides | Digitalis, Strophanthus, Scilla | Cardiotonic |
| 3. | Sinigrin (S-glycoside) | Mustard | Irritant property |
| 4. | Psorelen (Coumarin) | Bavchi | Leucoderma (white patches on skin) |
| 5. | Shatavarin-IV (Saponin) | Shatavari | Uterine Disease |
| 6. | Diosgenin (Saponin) | Dioscorea | Synthesis of sex hormone and oral contraceptive |
Volatile Oils
Introduction:
- The odorous, volatile principles of plant and animal sources are known as volatile oils. As they evaporate when exposed to air at ordinary temperatures, they are also called as “ethereal oils.”
- Volatile oils Contain terpenes, phenols, esters, and other aromatic molecules.
- They represent essence or active constituent of plant, or animal hence they are also known as “essential oils”.
- Volatile oils are soluble in alcohol, ether and other lipid solvents and practically insoluble in water. They are usually lighter than water. They possess characteristic odours and they have high refractive index. Most of them are optically active.
- They are excretory products produced during the metabolic processes of the plant, which are of no further importance in the process of metabolism.
Occurrence & Distribution:
- The volatile oils are widely distributed in plant kingdom more particularly among the phanerogams(bear seeds).
- Currently there are about 70 spices and 150 aromatic plant species cultivated in different parts of the world.
- In most cases oil exists in various organs of the plants such as leaves, flowers, fruits, stems or roots. They are found in glands or in canal like intercellular receptacles.
- Few of the volatile oils are formed after hydrolysis during the process of preparation, for example, bitter almond oil from amygdalin, or mustard oil from sinigrin.
- They are secreted in special structures such as duct, cell, schizogenous or lysigenous glands, trichomes, etc.
- They are commonly found in the species of Labiatae (Mint family), Rutaceae (Citrus family), Piperaceae (Pepper family), Zingiberaceae (Ginger family), Umbelliferae (Carrot family), Myrtaceae (Myrtle family), and Lauraceae (Laurel family).
General Methods of Isolation:
Volatile or essential oils are steam volatile constituents which are found in ducts, cavities or glandular hair of the plants. The most common method for the production of volatile oils is distillation. Here are the different extraction processes of raw materials that exist in perfumery.
- Water Distillation
- Steam Distillation
- Extraction with volatile solvents
- Expression
- Enfleurage
- Head space
- Extraction by CO2 or sofact
1. Water Distillation
- Process: Plant material is submerged in water and boiled. Steam carries volatile oil to a condenser where it cools and separates.
- Advantages: Simple and cost-effective. Suitable for hardy plant materials like roots and barks.
- Disadvantages: Not ideal for heat-sensitive oils, as prolonged heating may degrade them.
2. Steam Distillation
- Process: Steam is passed through plant material in a separate chamber. The steam volatilizes the oil, which is then condensed and collected.
- Advantages: Protects heat-sensitive oils by reducing direct contact with water. Widely used in the industry for flowers and leaves.
- Examples: Oils of lavender, peppermint, and eucalyptus are often extracted this way.
3. Extraction with volatile solvents
- Process: Solvent is passed through the plant material. Non-polar solvents like hexane or ethanol are used to dissolve the volatile oils. Solvent is then evaporated to leave a concentrated residue (concrete or absolute).
- Advantages: High yield and retains delicate aromatic compounds. Suitable for flowers like jasmine and rose.
- Disadvantages: Residual solvents may remain in the final product. Costly and complex.
4. Expression:
- Process: Physical pressing or squeezing of plant material (typically citrus peels). The oil is separated mechanically.
- Advantages: No heat involved, preserving oil integrity. Simple and effective for citrus oils (e.g., orange, lemon).
- Disadvantages: Limited to oil-rich materials like citrus peels.
5. Enfleurage:
- Process: Fresh flower petals are placed on glass sheets coated with odorless fat. The fat absorbs the aromatic compounds, and the oil is later extracted using alcohol.
- Advantages: Gentle method, ideal for delicate flowers like jasmine and tuberose.
- Disadvantages: Time-consuming and labor-intensive. High cost limits its use in modern industries.
6. Head Space:
- Process: Plant material is placed in a sealed chamber. Absorbing a flower\\\\\\\'s scent without physically altering the plant. Volatile compounds in the airspace above the material are collected and condensed.
- Advantages: Captures even the most fleeting aromatic compounds. Ideal for research and creating unique fragrances.
- Disadvantages: Requires specialized equipment. Expensive for large-scale use.
7. CO2 Extraction (Sofact):
- Process: CO₂ gas is pressurized into a supercritical fluid (between a liquid and gas state). This fluid dissolves volatile oils, which are separated by reducing pressure.
- Advantages: Produces pure, solvent-free oil. Ideal for heat-sensitive and high-value oils.
- Disadvantages: Expensive and requires sophisticated equipment.
Pharmaceutical Uses:
Volatile oils have a broad range of applications beyond perfumery and cosmetics. They are vital ingredients in the manufacture of soaps, toiletries, deodorizers, and are often used as masking agents in cleaning products, polishes, and insecticides.
Moreover, essential oils are valued in the pharmaceutical industry for their therapeutic properties. They possess carminative action and various therapeutic benefits, making them useful in medicinal formulations. Additionally, these oils find extensive use as natural flavors for foods and confections due to their aromatic and flavor-enhancing qualities, contributing to a wide array of consumer products.