UNIT 2.0 NATURAL PRODUCT CHEMISTRY

LEARNING OUTCOMESAt the end of the unit 2, students should be able to :v Describe the principle of natural products chemistry

v Explain compound involves in primary metabolites

v Describe function of secondary metabolites and example

v Differentiate the primary and secondary metabolites

v Explain steps and methods involved in natural products chemistry

2.1- INTRODUCTION

§ Chemistry of natural product deals with the chemistry of metabolites

What is Metabolites????§Metabolites are naturally occurring organic compounds synthesized by plants through metabolic activities in plants, aided by enzymes.

§Metabolites are the intermediates and products of metabolism.

The Metabolism is the sum of all the chemical reactions that take place in a living organism.

Plant metabolism :

complex of physical and chemical events of photosynthesis,respiration and the

synthesis and degradation of

organic compounds.

All the chemical products involved in thePrimary Metabolism– carbohydrates, protein, fatty acid, etc.

*Primary Metabolism: All the chemicalreactions necessary for the live of theorganism by itself.

All the chemical products that are producedby the living organism but are NOT involvedin the primary metabolism.

Not necessary for the live of the organism byitself but useful for interaction with others

(chemical communication, chemical defences).

PRIMARY METABOLITES SECONDARY METABOLITES

WITH and WITHOUT

1. INTRODUCTION TO THE CHEMISTRY OF NATURAL PRODUCTS 7

What are “Natural Products”? Chemical characteristics

• Naturally-occurring small organic compounds• including heterocyclic compounds, and peptides. • does not include proteins, carbohydrates, and nucleic

acids.• MW: ~150 ~ <800 amu (“small molecule”)• Methods of extraction and purification are generally similar to the techniques used for organic compounds

(e.g., TLC, column chromatography, HPLC, GC)• Methods of structural determination

• NMR, MS, IR, X-ray, UV

1. INTRODUCTION TO THE CHEMISTRY OF NATURAL PRODUCTS 8

What are “Natural Products”? Biological characteristicsØ Compounds are generally characterized of a particular

species or familyi.e. narrow taxonomic distribution

Ø No nutritional or structural function. Ø Functional roles may include:

- color (identification) - scent (attraction or repulsion)- sexual attraction - social communication- defense (e.g., plant toxins and antibiotics)

Ø but many still have unknown function in the organismØ Classified as “secondary metabolites” in contrast to

“primary metabolites”

UNIT 2.2PHYTOCHEMISTRY

2.2 PHYTOCHEMISTRY

Phytochemistry is the study of phytochemicals (chemicals derived from plants)

Phytochemical- chemical compounds that occur naturally in plants- act as natural defence mechanisms in their host plants (disease-preventing

compounds)- provide pigment (colour) and smell to the plants- Non essential nutrients, meaning that they are not required by the human

body for sustaining life

ü Blueberry is a fruit that is full of nutrients in both the flesh and skin.ü Skin contains a class of compounds called anthocyanidinsü compounds that give the blueberry its distinctive color

ü When we eat the berry, our body absorbs the nutritive factors (e.g.,sugars, vitamins, and nutritionally essential minerals), but we alsoconsume these non-nutritive anthocyanidins.

BLUEBERRY!!

ü Anthocyanidins is most widely known for their antioxidant properties.ü Antioxidants prevent the oxidization of certain compounds and fight

attacks on the body from harmful chemicals.ü The health benefits of antioxidants include anti-carcinogen qualities,

better heart health, and a range of other positive effects.ü Anthocyanidins in particular have been shown to have a positive effect

on collagen

BLUEBERRY!!

The following diagram illustrated the array of phytochemicals offered by a variety of fruits and vegetables.

PRIMARY

SECONDARY METABOLITES?

Primary MetabolitesPrimary metabolites are compounds that are commonly produced by all plantsand that are directly used in plant growth and development.

Component of primary pathways- enabling a plant to synthesize, assimilate anddegrade organic compounds

The main primary metabolites are carbohydrates, proteins, nucleic acids, andlipids.

Secondary MetabolitesØ more limited in distribution being found usually in specificfamilies.Ønot necessary for growth and development, but may serve aspollination attractants, environmental adaptations (defense), orprotection.

ATTRACT. DEFENSE. PROTECT

FUNTION OF SECONDARY METABOLITES

1) CarbohydratesØCarbohydrates are the sugars made up of glucose and its isomersØCarbohydrates come in many different sizes. Three subtypes are:ØMonosaccharides made up of one sugar unit (glucose or fructose)ØDisaccharides made up of two sugar units (sucrose is a glucose

and a fructose)ØPolysaccharides are polymers made up of more than two sugar

units - used to support plants

Compound in Primary Metabolites

Main functions

• Energy storage • Structure element• Source of carbon of the

biosynthesis of other substances

• “Markers” on cell surface for cell-cell recognition.

2) Proteinso Proteins make up most of the remaining biomass of living plantcells.o A protein consists of one or more polypeptides made up ofamino acids.o Plants make amino acids from the products of photosynthesisthrough a very complex process involving the acquisition of N,usually in the form of NH4, and involving the use of largeamounts of energy, in the form of ATP and NADPH.

Some functions of proteins

• Structural support eg collagen, keratin• Transport other substances eg haemoglobin• Movement eg the actin n myosin in muscles, protein in cilia and

flagella• Defense against foreign substances eg : antibodies combat

bacteria and viruses

3) Nucleic AcidsØ2 types of nucleic acids: DNA and RNA

Øpolymers of individual nucleotides : make up RNA and DNA. ØNucleotide has 3 components: ØFive-carbon-ring sugarØPhosphate groupØNitrogen base

ØNitrogen base: adenine, thymine/ uracil, guanine and cytosine

DNA RNAStands for Deoxyribonucleic Acid Ribonucleic Acid

Definition A nuclei acid that contains the genetic instruction used in the development and functioning of all modern living organisms. DNA’s genes are expressed, or manifested, through the proteins that its nucleotides produce with the help of RNA.

The information found in DNA determines which traits are to be created, activated or deactivated, while the various forms of RNA do the work.

Function The blueprint of biological guidelines that a living organism must follow to exist and remain functional. Medium of long term, stable storage and transmission of genetic information

Helps carry out DNA’s blueprintguidelines. Transfers genetic code needed for the creation of proteins from the nucleus to the ribosome.

q A broad category of chemical compounds, also referred as fat. qMost of those products are non polar fat, oil, or wax that does not (or poorly) dissolves in

water. qMain functions: Main component of cells membrane, energy storage, communication between

cells (hormones)

4) LIPIDS

PRIMARY METABOLITES SECONDARY METABOLITESessential to cell growth, and they are involved directly in metabolic reactions such as respiration and photosynthesis.

Not necessary for growth. Involved in defense reactions

are identical among most organisms numerous and wide spread

produced during the growth phase of cell

produced during the non- growth phase of the cell

accumulated by plant cells in largequantities

accumulated by plant cells in very small quantities

Ex : Proteins, carbohydrates, nucleic acid etc

Ex : alkaloids, phenolics, sterols, steroids and lignins etc

DIFFERENCES BETWEEN PRIMARY & SECONDARY METABOLITES

• FORM 4 GROUPS

• EACH GROUP WILL PRESENT A TOPICØ ALKALOIDSØ TERPENOIDØ FLAVONOIDSØ SAPONINS

ACTIVITY 2 : PRESENTATION ON SECONDARY METABOLITES

• PRESENTATION SHOULD DISCUSSØ DEFINITIONØ EXAMPLESØ STRUCTURESØ FUNCTIONS

• PRESENT ON TUESDAY, 16 JANUARY 2018

UNIT 2.3

METHODS IN NATURAL PRODUCTS CHEMISTRY

Identification of plant material and its active compound

Extraction (eg: cold extraction, Soxhlet extraction)

Isolation and purification (eg: partitioning, ion-exchange chromatography)

Structural elucidation (eg: Mass spectrometry, NMR spectroscopy)

ØIdentification and classification of plants are conducted by fieldbotanist (into a species and family); then the samples are collectedfrom aerial parts, the trunk bark and roots.Ø Identity of plant can be achieved by macro- and microscopicalexaminations.ØVoucher specimens are reliable reference sources to compare thecollected plant.ØThe collected samples are then gently air-dried : biomass must bedried quickly to avoid degradation of components by air / microbes.

IDENTIFICATION OF PLANT MATERIAL AND ITS ACTIVE COMPOUND

Drying of plant materials

Aim of drying: 1. Ease of transport 2. Ease of grinding3. Inhibit the growth of microorganisms 4. Preservative of active constituents

Plant may be collected from:

1 - Wild plants. 2 - Cultivated plants.

Wild plant Cultivated plant

Scattered in large or unlimited area

Present in limited area

Difficult to reach Easy to reachWild plant identification

need highly skilled botanists

Identification of cultivated plant is easier than wild

plantDeficiency may occur due to continuous collection

Continuous supply

The following precautions should be considered duringstage of collection:1. Proper time of the day, time of the year and maturity stage of

collection is particularly important because the nature andquantity of constituents may vary greatly in some species accordingto the season and time of collection

2. Collected plant should be free from any contamination.

3. Collected plants which are free from diseases

i.e. which are not affected by viral, bacterial, fungal infection

EXTRACTION

DEFINITIONEXTRACTION:

üThe separation of medicinally active portion of plants through the use of selective solvent and suitable methods extraction.

EXTRACT:

üa substance made by extracting a part of a raw material, often by using a solvent such as ethanol or water

EXTRACTION

There is no general (universal) method for the extraction of plantmaterials.

The precise mode of extraction depends on:1- The texture of the plant material. 2- The water content of the plant material.3- The type of substances to be extracted or nature of active constituents.

1) Maceration2) Percolation3) Infusion4) Decoction

PRINCIPAL METHODS OF EXTRACTION

5) Digestion6) Continuous hot extraction

(Soxhlet extraction process)7) Liquid-liquid extraction8) Distillation

Ø Powdered plant material is soaked in organic solvent and kept for about 24 hrs , sometimes 3-4 days also, depending upon the part of the plant to be extracted.

ØThe solvent is decanted, filtered and concentrated.

Method: MACERATION

Method: PERCOLATIONØThis method uses percolator

ØThe powdered material is packed in the percolator

ØEnough solvent is than poured to soak the powder

ØThe extract is collected in a receiver

ØThe process can be repeated as many times as necessary to ensure full extraction.

ØMore solvent is poured and allowed to percolate through the material.

ØThe process is continued until extraction is complete

ØPowdered plant material is boiled with the solvents.

ØIt is collected and filtered.

ØFiltered solvent is concentrated.

ØDecoction is only suitable for extracting heat-stable compounds and hard plants materials (e.g. roots and

barks)

ØEg. Preparation of tea.

Method: DECOCTION

Method: SOXHLET EXTRACTIONØMost widely used method for extraction of plantnatural products

ØThis is a continuous process of extraction with ahot solvent

ØThis extractor is provided with a siphoning system

ØPowdered plant material is packed in a thimble

ØSolvent is boiled in a flask

ØThe evaporated solvent passes through the side tube of the extractor and condensed in the condenser, pitted at the top of the extractor

ØThe condensed hot solvent runs into the thimble and soaks the material and extracts the constituents

ØWhen the chamber holding the thimblebecomes full, the solvent siphons down to the flask and the process is continuously repeated until complete

Successive solvent extraction example :

Petroleum ether (60-80)

Benzene

Chloroform

Acetone

Ethanol (95%)

Chloroform water

ISOLATION AND PURIFICATION

PLANT MATERIAL EXTRACT COMPOUND

Ø Extraction protocol ØActivity is demonstrated in a bioassayØfractionate the extract using a separation method so that a purified biologically active component can be isolated

Ø Identification

PARTITIONINGØPossibly simplest separation method

ØWidely used as an initial extract purification and ‘clean up’ step

ØUse two immiscible solvents to which the extract is added

ØA soft separation method – relies on the solubility of natural products and not a physical interaction with another medium

ØExample: Compounds that differ greatly in solubility such as monoterpenes separated from phenolics

CHROMATOGRAPHY

CLASSIFICATION OF CHROMATOGRAPHY Mobile phase

Packing of stationary phase

Force of separation

Classification according to mobile phase:

Classification according to packing of stationary phase:1 Thin Layer Chromatography (TLC) : the stationary phase is a thin layer of sorbents supported on glass, plastic or aluminum plates2 Paper chromatography (PC) : the stationary phase is a strip of chromatography paper3 Column chromatography (CC) : the stationary phase is packed in a glass column

Classification according to force of separation:

EXAMPLE OF CHROMATOGRAPHY

METHODS

THIN LAYER CHROMATOGRAPHY (TLC)

1 2 3 4 5 6 7 8 9 10 11 12

TLC CHROMATOGRAMS OF Orthosiphon stamineus

ØNatural products that are not UV-active need development using spray reagent such as vanillin-sulphuric acid, Dragendorff’s reagent, phosphomolybdic acid or antimony trichlorideØSeparated compound visualized as colouredbands

COLUMN CHROMATOGRAPHY (CG)

1. Concentration (heat and stir)

2. Slow evaporation

3. Refrigeration

Based on differences in solubility of the components of a mixture in a particular solvents

Methods of crystallization

ISOLATION AND PURIFICATION

STRUCTURAL ELUCIDATIONüStructure elucidation is the process of determining thechemical structure of a compound.üFor organic compounds, it will often involve the use ofnuclear magnetic resonance spectroscopy (NMRspectroscopy).üOther characterization techniques include massspectrometry, infrared spectroscopy and X-raycrystallography.

NUCLEAR MAGNETIC RESONANCE (NMR)Nuclear Magnetic Resonance (NMR) spectroscopy isØan analytical chemistry technique used in quality controland research to determine the content and purity of asample for as well as its molecular structure.Types of NMRØProton NMR

ØCarbon-13 NMR

INFRARED SPECTROSCOPYØInfrared Spectroscopy is the analysis of infrared light interacting with a molecule. ØThis can be analyzed in three ways by measuring absorption, emissionand reflection.

ØThe main use of this technique is in organic and inorganic chemistry. ØUsed by chemists to determine functional groups in molecules.

X-ray CrystallographyX-ray crystallography is a tool used for identifying the atomicand molecular structure of a crystal.

Øcrystalline atoms cause a beam of incident X-rays to diffractinto many specific directions.

Mass SpectrometryØA mass spectrometer can rapidly and accurately measure the molecular weight and quantities of many substances.

ØThis technique alone cannot distinguish between two or more substances with the same molecular weight.

LEARNING CHECK1- How plant material can be identified?2- State 4 common extraction methods to extract plant material.3- Name common methods/techniques used in isolation and purification.4- State the instruments used in structural elucidation.

THANK YOU