1. Pendahuluan, Metabolit Primer Dan Sekunder.pdf

Ika Putri Nurhayati Jurusan Farmasi FKUB

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Phytochemistry

A study of the chemical composition of plants

Explanation of the various plant processes in which chemical phenomena are concerned

- Qualitative detection of plant component - Actual isolation of plant component - Quatitative estimation of plant component 5

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Before the availability of synthetic drugs, phytodrugs or herbal drugs were the mainstay of treatment

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An analysis into the sources of new drugs from 1981 to 2007 reveals that almost half of the drugs approved since 1994 were based on natural products. During the years 2005–2007, 13 natural product related drugs were approved.

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Cancer and infections are the two predominant therapeutic areas for which the drug discovery program is based on natural products, but many other therapeutic areas also get covered, such as neuro-pharmacological, cardiovascular, gastrointestinal, inflammation, metabolic, etc 6

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PRIMARY METABOLITES • Compound involved in primary metabolism, in which demonstrate the fundamental unity of all living matter • In plants, primary metabolism is made up of photosynthesis, respiration, etc., using CO2, H2O, and NH3 as starting materials, and forming products such as glucose, amino acids, nucleic acids. These are similar among different species. • Essentially the same in all organism

SECONDARY METABOLITES

• In secondary metabolism, the biosynthetic steps, substrates and products are characteristic of families and species. Species which are taxonomically close display greater similarities (and metabolites); those which are distant have greater differences. • Not necessarily produced under all conditions • Ex. Flavonoid, alkaloid, terpenoid

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Primary • Sources of metabolic energy and energy transfer • Cellular building block and structural support, ex: lignin • Source of genetic information ex: nucleus, ribosom, ER • Catalyst of metabolic reaction

Secondary • Deterrence of predators and pathogens, ex: nicotine, hyociamine, menthol, cyanide glycoside • Attraction and deterrence of pollinators • Allelopathic action, ex: Arctostaphylos uva-ursi (bearberry) that inhibit the growth of grasses (Poaceae family) and herbs • Attraction of Symbionts, ex: host plant and rhizobia nitrogen-fixing bacteria (leguminoceae family 9

 In green plants,

carbohydrates are synthesized by the process of photosynthesis utilizing carbondioxide and water.  In most of the plants, sucrose is the major form of carbohydrates to be translocated across the plant body. Photosynthetically fixed carbon can also get stored in the form of starch.

Size of Carbohydrates : Monosaccharides made up of one sugar unit (glucose or fructose) Disaccharides made up of two sugar units (sucrose is a glucose and a fructose) Polysaccharides are polymers made up of more than two sugar units 10

Polysaccharides Structural polysaccharides are used to support plants

Storage polysaccharides are used to store energy for later use by the plant The most common structural polysaccharide in plants is cellulose. It makes up 40 to 60% of the cell wall. It is also the most common polymer on earth Cellulose is extremely strong due to its chemical organization. It is made of a long chain of betaglucose molecules – 100 to 15,000 glucose molecules

Storage Polysaccharides The most important storage polysaccharides are amylose and amylopectin. Amylose is a long chain of alpha-glucose, several hundred to several thousand molecules long. Amylopectin is more complex, often made up of 50,000 molecules.

These two polymers are both used in making starch grains. Most starch grains are about 20% amylose and 80% amylopectin, but this varies with the plant.

 Plant use fats for mainly for carbon storage, but also for energy production  lipids, which make up both the plasma membrane and the membranes of all internal compartments and organelles;  Most lipids have a fatty acid portion made from acetyl-CoA and malonyl-CoA in a reaction whose repetition produces longer molecules.

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OILS Oils are fats that are liquid at room temperature. Oils occur in all parts of a plant, but are most common in seeds. Some seeds have so much oil that it can be commercially harvested. The most commonly used oils are cotton, sesame, safflower, sunflower, olive, coconut, peanut, corn, castor bean, and soybean oils.

The most common seed oil fatty acids are oleic acid (one double bond), linoleic acid (two double bonds), and linolenic acid (three double bonds). Linoleic and linolenic are essential fatty acids – we can’t make them ourselves.

WAXES Waxes are complex mixtures of fatty acids linked to long-chain alcohols. Waxes comprise the outermost layer of leaves, fruits, and herbaceous stems and are called EPICUTICULAR waxes. Waxes embedded in the cuticle of the plant are cuticular waxes. Cutin is another wax in the cuticle and it makes up most of the cuticle. Suberin is a similar wax that is found in cork cells in bark and in plant roots. Both help prevent water loss by the plant. Structures of waxes vary depending on which plant produced them.

Waxes are usually harder and more water repellant than other fats.

 proteins, which make up both structural units of the cell such as microtubles and all the enzymes of every biochemical process  nucleic acids and nucleotides, which code for all proteins, act as metabolic energy molecules such as ATP  Ribosomes, for example, consist of both protein and RNA, the combination of which allows the production of all other proteins.

enzymes Enzymes catalyze biochemical reactions. Most proteins in living cells are enzymes. Pure enzymes that maintain their activity when removed from plants are commercially important to us.

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Secondary metabolites defend plant againts a variety of herbivores and pathogenic microbes. SM may serve other imprtant functions as well, such as structural support (ex lignin) or pigmen (ex anthocyanin) Secondary metabolites or secondary compounds are compounds that are not required for normal growth and development, and are not made through metabolic pathways common to all plants. Plant secondary metabolites can be divided into three chemically distinc groups  terpenes, phenolics, and nitrogen containing compound The building blocks for secondary metabolites are derived from primary metabolism 17

Chemical Class Monoterpenes

Terpenoids

Sesquiterpenes

Largest group with structures identified, ~30,000 compounds

Diterpenes

Compound Example Essential oils and volatiles (e.g. menthol)

Triterpenes

Plant Examples Mint

Cucumber

Tetraterpenoids

Phenolics More ubiquitous in terms of presence in plants

Tannins

Condensed tannins

Oaks

Lignins Flavonoids

Nitrogen Containing

Alkaloids

Nicotine, Cocaine

Tobacco

Nitrogen and Sulfur Containing

Glucosinolates

Sinigrin

Cabbage and other Brassicaceae

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 Plant produce a large variety of secondary products that contain a phenol group, classified as phenolic compound.  Plant phenolic compound are chemically heterogenous groups of nearly 10.000 individual compound.  Phenolics plays a variety roles in the plant, such as defense compound, mechanical supporty, attracting pollinators and fruit dispersers, absorbing harmful ultraviolet radiation, reducing the growth of nearby competing plants.

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 Two basic pathways in phenolic biosynthesis are shikimic acid pathway and

the malonic acid pathway. The malonic pathway play important role in phenolic production in fungi and bacteris, but less significance in higher plant.  The shikimic acid pathways converts simple carbohydrate precursors derived from glycolysis and the pentose phosphate pathways to the aromatic amino acid. This pathways present in plant, fungi, and bacteria, but is not found in animals.  Mostly, phenolic compound in plants are derived from phenylalanine via the elimination of an ammonia molecule o form cinnamic acid.  Trans-cinnamic acid, p-coumaric acid, and their derivatives are simple phenolic compounds called phenylpropanoids because they contain a benzene ring and three-carbon side chain. Phenylpropanoid are important building blocks of the more complex phenolic compound. 21

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 Simple phenylpropanoid and benzoic acid, such as caffeic acid and ferulic acid occur in soil in appreciable amounts and have been shown to inhibit the germination and growth of many plants.  Lignin, polymer of phenylpropanoid, is found in the cell walls of various types of supporting and conducting tissues providing mechanical support and protective function in plants.  Flavonoid are one of the largest classes of plant phenolics. The basic carbon skeleton of flavonoid contain 15 carbons arranged in two aromatic rings connected by a three-carbon bridge. Isoflavonoids are group of flavonoids in which the position of a one aromatic rings (ring B) is shifted.  A second catergory of plant phenolic polymers beside lignin, is tannin. There are two categories of tannin condensed and hydrolyzable.

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 The basic structural elements of terpenese are sometimes called isoprene units because terpenes can decompose at high temperature to give isoprene  Terpenese are classified by the number of five carbon unit they contains. Ten carbon terpenes which contain two C5 unit called monoterpenes, 15 C (three C5 units) are sesquiterpenes, 20C (four C5 unti) are diterpenes, 30 C are triterpenes  Terpenes are biosynthesized from primary metabolites in at least two different ways, mevalonic pathways and methylerythriol phosphate (MEP) pathway

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 The red, orange, and yellow carotenoids are tetraterpenes that function as    

accessory pigment in photosynthesis. In conifers such as pine and fir, monoterpenes accumulate in resin ducts found in the needles, twigs, and trunk. These compound are toxic to numerous insect. Many plants contain mixtures of volatile monoterpenes and sesquiterpenes, called essential oils, that lend characteristic odor to their foliage. Mono and sesquiterpenes are commonly found in gandular hair on the plant surface, especially the terpenes are stored in a modified extracellular space in the cell wall. Essential oil have wellknown insect repellant properties. 26

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 Plant secondary metabolites that contain nitrogen in their

structure are alkaloid, cyanogenic glycosides, glucosinolates, etc. Most nitrogenous secondary metabolites are biosynthesized from common amino acids.  Nitrogen atom in alkaloid is usually part of a heterocyclic ring, that contain both nitrogen and carbon atoms.  Most alkaloid are alkaline. At pH values commonly found in the cytosol (pH 7,2) or the vacuole (pH 5 to 6), the nitrogen is protonated, hence alkaloid are positively charged and are generally water soluble.  Most alkaloid are now believed to function as defense againts predators because of their general toxicity and deterence capability. 28

 Cyanogenic glycosides and glucosinolates are not themselves toxic but are

readily broken down to give off volatile poisons when plant is crushed.  Cyanogenic glycosides release the well-known poisonous gas hydrogen cyanide (HCN).  Cyanogenic glycosides are not normally broken down in the intact plant because the glycoside and the degradative enzymes are spatially separated, in different cellular compartements or in the different tissues.  Cyanogenic glycosides have protective function in certain plants. This deters feeding by insect and other herbivores, such as snails and slugs.  Glucosinolates found principally in the Brassicaceae and related plant families. These compound responsible for the smell and taste of vegetables such as cabbage, broccoli, and radishes.  Glucosinolates break down to release volatile defensive substances. 29

 Alkaloid are usually synthesized from one of a few common

amino acids, in particular lysine, tyrosinen, and tryptophan.  Carbon skeleton of some alkaloid contains a component derived from the terpene pathways  Nearly all alkaloid are also toxic to humans when taken in sufficient quantity. For example strychnine, atropine, and coniin are classic alkaloid poisoning agents. At lower doses, many are useful pharmacologically.  Morphine, codeine, and scopolamine are just a few of the plant alkaloid currently used in medicine. Other alkaloid, including cocaine, nicotine, and caffeine, enjoy widespread nonmedical use as stimulant or sedatives. 30

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PHYTOCHEMICAL SCREENING Phytochemical screening  The plant extracts analyzed for the presence of secondary metabolites like alkaloids, terpenes, and flavonoids. Following this, a simple separation technique like thin-layer chromatography (TLC) is generally used to analyze the number and type of components present in the mixture.

The tests are simple to perform, however, it is not suitable for the efficient separation of metabolites, and has low selectivity and sensitivity of detection. This procedure enables the early recognition of known metabolites in the extracts, and is thus economically viable. 33

PURPOSES OF PHYTOCHEMICAL SCREENING

Identify phytochemical group of new explored plants

Finding chemical constituents in the plant that may lead to their quantitative estimation

Locating the source of pharmacologically active compound

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METHODS Should give additional information as to the presence or absence of specific members of the group being evaluated

Simple Rapid

A method for use in phytochemical screening should be

Quantitative in so far as having a knowledge of the lower limit of detection is concerned, and if possible

Designed for a minimum of equipment

Reasonably selective for the class of compounds under study 35

DETECTION OF ALKALOID Alkaloids usually occur in plants as their water-soluble salts

Most alkaloids are precipitated from neutral or slightly acid solution by  Mayer's reagent (potassio mercuric iodide solution)  Cream precipitates  Wagner's reagent(solution of iodine in potassium iodide)  Reddish-brown precipitates  Hager's reagent (a saturated solution of picric acid)  yellow precipitates  Dragendorff's reagent (solutionof potassium bismuth iodide)  Reddish-brown precipitates Care must be taken in the application of these alkaloidal tests, as the reagents also give precipitates with proteins 36

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ALKALOID SCREENING USING TLC

All Rauwolfia alkaloids give with Dragendorff reagent orange-brown zones

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DETECTION OF FLAVONOID Flavonoid aglycone polyphenols are slightly acidic and will thus dissolved in alkali.

in the presence of oxygen many flavonoid will degrade

presence of sugar tends to render the flavonoid more H2O soluble

generally dissolved in polar solvent such as methanol, ethanol, acetone, dimethyl sulfoxamide,dimethly furomide, water etc

while polar aglycones such as isoflavone flavonone and slightly methoxylated flavone and flavonones tends to be more soluble in solvent such as ether and propanol 40

DETECTION OF FLAVONOID Alkaline reagent test: The Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colour less, on addition of few drop of dilute acid indicates the presence of flavanoids.

Lead acetate Test: The Extracts were treated with few drops of Lead acetate solution. Formation of yellow colour precipitate indicates the presence of flavanoids.

Ferric chloride test: 1 ml of the extract was treated with 1 ml of ferric chloride.Formation of brown colour precipitates indicates the presence of flavonoids 41

1. Alkaline reagent test

2. Lead acetate Test

3. Ferric chloride test

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FLAVONOID SCREENING

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FLAVONOID SCREENING

apigenin fluorescent spots when observed at 254 nm after derivatisation with NPPEG reagent.

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DETECTION OF SAPONIN Foam test: 0.5 gm of extract was shaken with 2 ml of water. It foam produced persists for ten minutes it indicates the presence of saponins.

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DETECTION OF TANNIN Gelatin Test: To the extract, 1% gelatine solution containing sodium chloride was added. Formation of white precipitate indicates the presence of Tannins.

Modified Prussian blue test: To 1 ml of extract, add 1 ml .008M potassium ferric cyanide and 1 ml of .oo2 M ferric chloride in 0.01 M HCl. Presence of blue colour indicates the presence of tannins.

Lead acetate Test: To the extract few drops of Aqueous basic lead acetate solution were added, reddish brown bulky precipitate indicates the presence of tannin.

Ferric chloride Test: To the extract, few drops of 1%natural ferric chloride solution was added formation of blackish blue colour indicates the presence of tannins

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DETECTION OF ANTRAQUINONE Borutrager’s Test: To 1 ml of the extract , add 1 ml of 10 % ferric chloride and .5 ml of concentrate hydrochloric acid. Boil in a water bath for few minutes. Filter it and the filtrate is treated with 1 ml of Diethyl ether and concentrate ammonia. Appearance of pink or deep red colour.

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Rhei radix analysis All aglycones show fluorescence quenching in W-254nm and uniformly yellow or orange-brown fluorescence in UV-365 nm. T1 rhein T3 emodin

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DETECTION OF CARDIAC GLYCOSIDES Keller-kiliani test 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. Reddish brown layer is formed, which turns bluish green after standing due to presence of digitoxose.

Legal test 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. Formation of pink colour in presence of glycosides or aglycon moiety

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DETECTION OF TERPENOID AND STEROID Libermann burchard test

• 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. Formation of violet to blue coloured ring at the junction of two liquid, indicate the presence of steroid moiety

Salkowaski test

• Alcoholic extract of drug was evaporated to dryness and extracted with CHCl3, add conc. H2SO4 from sidewall of test tube to the CHCl3 extract. Formation of yellow coloured ring at the junction of two liquid, which turns red after 2 min, indicate the presence of steroid moiety.

Antimony trichloride test

• Alcoholic extract of drug was evaporated to dryness and extracted with CHCl3, add saturated solution of SbCl3 in CHCl3 containing 20% acetic anhydride. Formation of pink colour on heating indicates presence of steroids and triterpenoids.

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Chromatography

• is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium.

mobile phase • a solvent that flows through the supporting medium

stationary phase • a layer or coating on the supporting medium that interacts with the analytes

supporting medium • a solid surface on which the stationary phase is bound or coated

Principles of Chromatography

Chromatogr aphy is a physical process.

We can only control stationary and mobile phase and the mixtures are the problem we have to deal with. Chromatography is a dynamic process in which the mobile phase moves in definite direction.

What are the OBJECTIVES of chromatography ?

To separate the components of mixture (preparative chromatography)

To identify the component of an unknown (analytical chromatography)

How does chromatography WORKS?

works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile phase.

molecules that spend most of their time in the mobile phase are carried along faster

Classification of Chromatographic methods According to mechanism

of separation:

depends mainly on the nature of the stationary phase. Chromatography can be classified into:

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Adsorpsion chromatography Partition chromatography Ion exchange chromatography Molecular/Size exclusion chromatography Zone electrophoresis Affinity chromatography Chiral chromatography

Adsorption Chromatography Separation is based on the interaction of the molecule in a mobile phase with the surface of the solid stationary phase (a liquid-solid interaction) This is a distribution process where the molecule interacts reversibly (binds and unbinds) with the surface molecules of the stationary phase (usually a finely divided solid)

Adsorption Chromatography The molecule and the mobile phase compete for adsorptive sites on the stationary phase. 1) Molecules (in the sample) that are most soluble in the mobile phase, will pass through the system quickly; 2) Molecules (in the sample) that are most soluble in the stationary phase will elute off the column slowly. Generally, the adsorbant is applied or bonded to a solid support (glass plate, plastic sheet) 1) Polar adsorbants : silica, alumina 2) Non-polar adsorbants : charcoals, paraffin

Partition Chromatography Separation is based on the interaction of the molecule in a mobile phase with the liquid stationary phase (a liquidliquid or gas-liquid interaction)

Separation of solutes is based on their differences in solubility between two immiscible phases (solvents). 1) The solubility of the molecules is based upon solute polarity and the concept of “like dissolves like” 2) The molecules (solute) of interest are ‘extracted’ into a solution based on relative solubilities

Partition Chromatography

normal phase

reverse phase

Ion exchange Chromatography Sample ions compete with the mobile phase ions for ionic sites on the stationary phase (charged functional groups) The sample ions that interact weakly with the stationary phase will be retained the least, whereas, those that interact strongly with the stationary phase will be retained the longest and will elute off the column later. Cation-exchange and anionexchange methods are available depending on the molecules to be separated

Ion exchange Chromatography

Molecular/Size exclusion Chromatography Separation is based on the size of the molecule Small molecules will elute off the column slowly Large molecules will elute off the column quickly

Zone Electrophoresis Chromatography

Separation based on size and charge Smaller molecules will migrate further, less tangled

Zone Electrophoresis Chromatography Sample application

Bands of postivey charged compounds

Bands of negativey charged compounds

Affinity Chromatography Very selective Specific binding site is used to concentrate analyte on column Used a lot in biological applications

It uses the affinity of proteins to specific ligands such as enzymes. The ligand is attached to suitable polysaccharide polymer such as cellulose - agarose – dextran.

Affinity Chromatography

Chiral Chromatography

In this type we can separate enantiomers – we used chiral stationary phase that react with one enantiomer more then the other so separation takes place.

CONCEPT OF POLARITY The interaction of attractive forces (molecular interactions) that exist between molecules is the basis of polarity Polarity is a relative term applied to solvents, solutes and adsorbents.

The degree of polarity is dependent upon the compound’s molecular geometry, electron configuration and its hydrogen bonding

CONCEPT OF POLARITY Organic solvents are generally non-polar; molecules are rigid and symmetrical; termed hydrophobic because they are not readily soluble in water (hexane, chloroform) H H H H H H – C – C – C – C – C – H (non-polar organic) H H H H H

CONCEPT OF POLARITY Alcohols and acids are generally polar compounds; molecules tend to be less symmetric and not as rigid; more soluble in water as compared to the organic solvents; termed hydrophilic because they are readily soluble in water (acetonitrile, isopropanol, HCl, H) H H H H H H – C – C – C – C – C – OH (polar alcohol) H H H H H

H H O H – C – C – C (more polar acid) H H OH

According to mobile phase: Chromatography can be classified into: 1- Liquid Chromatography (LC): The mobile phase is liquid. -Liquid-Solid Chromatography (LSC) Separation by adsorption, the stationary phase is solid -Liquid -Liquid Chromatography (LLC) Separation occurs through partition, the stationary phase is liquid

2- Gas Chromatography (GC) The mobile phase is inert gas nitrogen or helium. -Gas–Solid Chromatography (GSC) the stationary phase is solid -Gas-Liquid Chromatography (GLC) the stationary phase is liquid

According to technique (methods of holding the stationary phase): Chromatography can be classified into:

Column chromatography

• the stationary phase is contained in a tube called the column

Planar chromatography

• stationary phase is configured as a thin two-dimensional sheet

PLANAR CHROMATOGRAPHY

In paper chromatography a sheet or a narrow strip of paper serves as the stationary phase

In thin-layer chromatography a thin film of a stationary phase of solid particles bound together for mechanical strength with a binder, such as calcium sulfate, is coated on a glass plate or plastic or metal sheet.

PLANAR CHROMATOGRAPHY

COLUMN CHROMATOGRAPHY The primary division of chromatographic techniques is based on the type of mobile phase used in the system

Type of Type of Mobile Chromatography Phase Gas gas chromatography (GC) Liquid liquid chromatograph (LC)

According to purposeof use Chromatography can be classified into:

1- Analytical Chromatography: a. Qualitative Chromatography b. Quantitative Chromatography 2- Preparative Chromatography

Qualitative Chromatography 1- Confirm the absence or probable presence of certain constituent in the sample under investigation

2- Give an idea about the complexity of the mixture least number of compounds present.

and the

3- Check purity and identity of any compound.

4- Establish a (finger print ) pattern for extracts, volatile oils or pharmaceutical preparations. These finger prints can be then used to check the identity and purity in the future.

5- Monitor both column chromatography and organic chemical reactions.

Quantities Chromatography: The development of modern instruments enable the use of chromatography to determine the amount of any component in a mixture as absolute amount or relative to another component HPLC/ GC/ HPTLC can be used for there applications.

Preparatives Chromatography: This was the first and is the main application of chromatography. The technique was developed primarily for this purpose. Chromatography is used to obtain reasonable quantities of pure compounds from mixtures.

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