Introduction of natural products

What is natural product???

 Introduction to Natural Products
q Natural products are products from various natural sources, plants,
microbes and animals.
q Natural products can be an entire organism (e.g. a plant, an animal or a
micro-organism), a part of an organism (e.g. leaves or flowers of a plant, an
isolated animal organ), an extract of an organism or part of an organism and
an exudate, or pure compound (e.g. alkaloids, coumarins, flavonoids,
lignans, steroids and terpenoids) isolated from plants, animals or micro-
organisms.
q However, in practice, the term natural product refers to secondary

metabolites, small molecules (molecular weight < 1500 amu), produced by an

2
organism, but not strictly necessary for the survival of the organism.
 Chemical characteristics

v What are “Natural Products”?

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
 Biological characteristics

vWhat are “Natural Products”?

• Compounds are generally characteristic of a particular
species or family, i.e., narrow taxonomic distribution (non-
ubiquitous)
• No nutritional or structural function. Functional roles may
include:
• - color (identification) - scent (attraction or repulsion)
• - sexual attraction - social communication
• - defense (e.g., plant toxins and antibiotics)
• but many still have unknown function in the organism in which they are
found.
• Classified as “secondary metabolites” in contrast to “primary
metabolites”
 Introduction
Structural determination
Diverse aspects of the of natural products
compounds
Chemistry of Natural
Products: •Total synthesis or semi-
synthesis of natural products;
enzyme
Agricultural science:
Functional foods, herbal
antipest, allelopathy,
medicines
IPM
Biodiversity and Ecology; Chemotaxonomy and
Marine natural products genetic classification
synthesis
•Determination of biosynthetic
Pharmaceutical science:
pathways using using plant
tissues, cell culture and isotopic pharmacologic effects
labeling
Plant chemistry and
Ethnobotany
plant development
Genomics and
metabolonomics
 Why study Natural Products?

• Natural products are the source of the most complex and

fascinating chemical structures.
• Natural products represent biological diversity.
• Natural products are expressions of the genome.
• Natural products represent natural biological activity, whether as
single compounds or as complex mixtures.
• Natural products are part of the natural wealth of the country,
and can be an important source of livelihood, from agriculture
and food, pharmaceuticals, fine chemicals industry.
• Natural products can be an effective bridge from tradition to
modern scientific developments, including genetics, molecular
biology, biotechnology, and pharmaceutical science.
Range of products from natural
products
Range of products from natural products
 Do u have any idea on what
natural product produce that
u have ever seen in your
hometown?
 Introduction

The Changing Landscape of Herbal Medicine,

Food and Wellness
• Herbal Medicine
• Dietary Supplement
• Nutriceutical
• Functional Food
þ Natural Products Chemistry
is key to all of these!
 Natural products and Ecology

Animals
Antifeedant
Toxins
Toxins
Scents
Antipest
Attractants Toxins
Allelopathy Plants Insects Pheromones
Communication Communication

Antifungals
Symbiosis Symbiosis

Antifungals Chemotaxis
Chemotaxis Fungi Bacteria Communication
Communication Antibacterials
 Text

Text
Text
At the end of the 18th century, crude drugs were still being used as powders, simple extracts,
or tinctures.

The era of pure compounds (In 1803, a new era in the history of medicine)

Isolation of morphine from opium

Strychnine (1817)

Quinine and caffeine (1820) morphine

Nicotine Strychnine
Nicotine (1828)
caffeine
Atropine (1833)

Cocaine (1855)

Quinine
Atropine Cocaine
3
 In the 19th century, the chemical structures of many of the isolated compounds
were determined

In the 20th century, the discovery of important drugs from the animal kingdom, particularly
hormones and vitamins. In addition, microorganisms have become a very important source of
drugs

Compounds from natural sources play significant roles in modern medicine:

1. They provide a number of extremely useful drugs that are difficult, if not impossible, to
produce commercially by synthetic means
2. Natural sources also supply basic compounds that may be modified slightly to render
them more effective or less toxic
3. Their utility as prototypes or models for synthetic drugs possessing physiologic
activities similar to the originals

4 Aspirin Ibuprofen
 Overview of a bioassay-guided traditional natural product drug discovery process

Source materials Bioassay

Extract (s) Active extract (s)
(e.g. plant) Extraction
Chromatographic
fractionation

Bioassay Chromatographic
Active fraction (s)
fractions

Isolation and purification

Bioassay
Isolated compounds Active compound (s)

Identification by
spectroscopic techniques,
e.g. UV, IR, MS, NMR
Bioassay
5 Identified compounds Identified bioactive compound (s)
Are synthetic chemicals different
from natural ones?
• Synthetic chemicals are made by the use of
chemically reactive reagents. The chemicals tend to
fairly crude in bring about changes to structures –
addition, subtraction, substitution and
rearrangements

• Natural Products are made by enzymes which can be

much more selective – they can target their action on
parts of the molecule by bringing the active site of
the enzyme into close proximity with one part of the
molecule to be changed
The basic shape of Natural ProductMetabolism
primary metabolites
 Primary Metabolites
• Primary metabolites are compounds that are
commonly produced by all plants and that are
directly used in plant growth and development.
• Molecules that are essential for growthand
development of an organism.
• The main primary metabolites are: carbohydrates,
proteins, nucleic acids, and lipids.
• Some secondary metabolites are producedfor
easily appreciated reasons, e.g.
1. As toxic material providing defense againstpredators.
2. As volatile attractants towards the same or other
species.
3. As coloring agents to attract or warn other species.

• Secondary metabolism (º Natural products

chemistry).
• Secondary metabolites, are found in only
specific organisms, or groups of organisms, and
are an expression of the individuality of
species.

• Secondary metabolites are not necessarily

produced under all conditions, and in the vast
majority of cases the function of these
compoundsand their benefit to the organism
is not yetknown.
• How to differentiate between Primary and
Secondary metabolite?

• Search in the internet the success stories of

Primary and Secondary metabolite.

• Share your findings in the next class.

Ladybirds secrete alkaloid (coccinelline)-rich fluid (reflex blood) from leg joints as a defence
mechanism against predators. A technique is described that enables the collection and
accurate quantification of reflex blood produced, and the amount of coccinelline therein.
Coccinelline was found distributed throughout the body, although concentrated in the reflex
blood. Reflex blood was collected from a large set of beetles at several time points. Significant
variation was found among beetles in the amount of reflex blood produced (for males and for
females corrected for body weight) and the coccinelline concentration of the reflex blood
Over 300,000 secondary
metabolites in plants
Why study Natural Products?
•Natural products are the source of the most complex and
fascinating chemical structures.
• Natural products represent biological diversity.
• Natural products are expressions of the genome.
•Natural products represent natural biological activity, whether
as single compounds or as complex mixtures.
•Natural products are part of the natural wealth of the country,
and can be an important source of livelihood, from agriculture
and food, pharmaceuticals, fine chemicals industry.
•Natural products can be an effective bridge from tradition to
modern scientific developments, including genetics, molecular
biology, biotechnology, and pharmaceutical science.
The market for natural products is HUGE
• Pharmaceuticals
• Traditional herbal medicines:
US and Europe: gingko biloba, St. John’s wort, ginseng, garlic*,
echinacea, saw palmetto, soya*, kava-kava, golden seal, aloe*, gotu
kola* (*also grown in the Philippines)
India, China, Japan: Ayurverda, TCM, Kampo
Philippines: lagundi, sambong, ampalaya, banaba, malunggay
• Beverages: tea (e.g., green, chinese), herbal teas, coffee
• Food supplements and health products
• Fats and oils
• Herbs and spices, food flavor ingredients
• Perfumes and scents
• Essential oils, others …
 The study of natural products is multidisciplinary

Biology ¬ ® Chemistry

Taxonomy . Botany Organic synthesis

Agriculture . Pharmacology . Pharmaceutical Science . Organic analytical chemistry

Entomology . Microbiology . Biotechnology/Molecular Biology . Biochemistry

Genomics .

Proteomics

Metabolonomics
 Metabonomics offers
the opportunity to find
patterns of changes in the
entire metabolism.
(Donald Nicholson,
©International Union of
Biochemistry & Molecular Biology
http://pubs.acs.org/cen)
 Natural Products in History
• Natural products (secondary metabolites) have been the most successful source of potential drug
• Leads
• Natural products continue to provide unique structural diversity in comparison to standard combinatorial
chemistry, which presents opportunities for discovering mainly novel low molecular weight lead
compounds.
• Less than 10% of the world’s biodiversity has been evaluated for potential biological activity, many more
useful
• natural lead compounds await discovery with the challenge being how to access this natural chemical
• diversity.
• The earliest records of natural products were depicted on clay tablets in cuneiform from Mesopotamia
(2600 B.C.) which documented oils from Cupressus sempervirens (Cypress) and Commiphora species
(myrrh) which are still used today to treat coughs, colds and inflammation.
• The Ebers Papyrus (2900 B.C.) is an Egyptian pharmaceutical record, which documents over 700 plant-
based drugs ranging from gargles, pills, infusions, to ointments.
• The Chinese Materia Medica (1100 B.C.) (Wu Shi Er Bing Fang, contains 52 prescriptions), Shennong Herbal
(~100 B.C., 365 drugs) and the Tang Herbal (659 A.D., 850 drugs) are documented records of the uses of
natural products
• The Greek physician, Dioscorides, (100 A.D.), recorded the collection, storage and the uses of medicinal
herbs, whilst the Greek philosopher and natural scientist, Theophrastus (~300 B.C.) dealt with medicinal
herbs.
• During the Dark and Middle Ages the monasteries in England, Ireland, France and Germany preserved this
Western knowledge whilst the Arabs preserved the Greco-Roman knowledge and expanded the uses of
their own resources, together with Chinese and Indian herbs unfamiliar to the Greco-Roman world [3].
• It was the Arabs who were the first to privately own pharmacies (8th century) with Avicenna, a Persian
pharmacist, physician, philosopher and poet, contributing much to the sciences of pharmacy and medicine
through works such as the Canon Medicinae.
Early investigations of natural
products

In the so-called pre-scientific era

• Natural products having a history as folk
remedies were in use. Fore examples, opium,
belladonna, cinchona bark, etc. Many drugs
originally used as folk remedies, nowadays,
have been abandoned.
In the late eighteenth and early nineteenth centuries,
chemical experimentation led ultimately to its use in the
discovery of new drugs.
• In 1853, Henry How conceived the idea that
functional groups in natural products might be
modified by chemical reagents.
He heated morphine with methyl iodide, hoping to
convert the alkaloid to codeine. He obtained, however, a
new substance of the quaternary salt of morphine.

HO O
H OH
• In 1898, the first commercially available
semisynthetic morphine derivative (ethyl ether)
was introduced as a cough sedative in preference
to codeine or other opiates.
• Meanwhile, diacetylmorphine was introduced as a
safer pain reliever than morphine. It quickly
became popular throughout the world.

• Four years passed before its addictive properties of heroin

were recognized. Laws were later passed by governments
to restrict its use.
Developments of Medicinal Chemsitry Leading to
Various Medicinal Classes of Drugs
• During the 1840s, the first use of synthetic
organic chemicals were introduced for
anesthesia during a tooth removal, such as
nitrous oxide, ether, and chloroform.
• In 1864, barbituric acid had been synthesized as
a useful hypnotic.
• In 1875, salicylic acid was introduced as a
possible cure for typhoid fever. It was found to
be an effective antipyretic.
• In 1899, Aspirin was marketed as an antipyretic
without the unpleasant side effects. This
indicated that the chemical structures from
natural products were changed into better drugs.
• Medicinal Chemistry began.
Fast Development from 1900’s to
1960’s
• 1920’s~1930’s: Anesthetics, Hypnotics, Analgesics were
used extensively. In research for functional
“pharmacophore”, structure-function relationship was
investigated gradually.
• After 1930’s: The development of new drugs was speeded
greatly by the close combination of Medicinal Chemistry
and Experimental Pharmacology.
• Theory of antimetabolite was formed by using metabolic
products as lead compounds.
• Discovery of penicillin which is the first antibiotics is an
epoch-making achievement.
• Afterward, tetracycline, streptomycin, chloramphenicol, erythromycin
were introduced one after another.
• In 1940’s, the first drug used for treating cancer
as a biological alkylating agent was nitrogen
mustard, which began tumor chemical therapy.
• In 1960’s, oral steroidal contraceptive agents
were discovered. Corticosteroids have become
an important drugs.
• After 1950’s, aging disease, cerebrovascular and
cardiovascular diseases became first reason for
human death. New drugs design based on
agent,
enzymes or receptors as drug targets.
• In 1964, first β-Adrenergic blocking
Propranolol, was marketed.
• In 1979, Nifedipine, Calcium Channel Blocker was
marketed.
• In 1981, Captopril, Angiotensin Converting Enzyme
(ACE) Inhibitor was launched.
 Medicinal Plants inFolklore

• The use of natural products as medicines has been described throughout history
in the form of traditional medicines, remedies, potions and oils with many of
these bioactive natural products still beingunidentified.
• The dominant source of knowledge of natural product uses from medicinal plants
is a result of man experimenting by trial and error for hundreds of centuries
through palatability trials or untimely deaths, searching for available foods for the
treatment of diseases
• One example involves the plant genus Salvia which grows throughout the
southwestern region of the United States as well as northwestern Mexico and
which was used by Indian tribes of southern California as an aid in childbirth.
Male newborn babies were “cooked” in the hot Salvia ashes as it was believed
that these babies consistently grew to be the strongest and healthiest members
of their respective tribes and are claimed to have been immune from all
respiratory ailments for life.
 Medicinal Plants inFolklore

• The plant, Alhagi maurorum Medik (Camels thorn) secretes a sweet, gummy material
from the stems and leaves during hot days. This gummy sap is called “manna” and
consists mostly ofmelezitose, sucrose and invert sugar and it has been documented and
claimed by the Ayurvedic people that the plant aids in the treatment of anorexia,
constipation, dermatosis, epistaxis, fever, leprosy, and obesity. It was also used by the
Israelis who boiled the roots and drank the extract as it stopped bloody diarrhea. The
Konkani people smoked the plant for the treatment of asthma, whilst the Romans used
the plant for nasal polyps.
• The plant Ligusticum scoticum Linnaeus found in Northern Europe and Eastern North
America was eaten raw first thing in the morning and was believed to protect a person
from daily infection; the root was a cure for flatulence, an aphrodisiac and was used as a
sedative in the FaeroeIslands.
• Atropa belladonna Linnaeus (deadly nightshade) is found in central and Southern
Europe, Western Asia, North Africa, North America and New Zealand. Its notoriously
poisonous nature (three berries are sufficient to kill a child) firmly excluded it from the
folk medicine compilation and seemed to have been accepted as dangerous to handle
or to experimentwith.
 Historically Important Natural Products

Traditional medicinal practices have formed the basis of most of the early medicines followed
by subsequent clinical, pharmacological and chemicalstudies.

• Salix alba L. (willow tree) - acetylsalicyclic acid (aspirin) - anti-inflammatoryagent

• Papaver somniferum L. (opium poppy) – morphine (alkaloids) - painkiller.
• Digitalis purpurea L. (foxglove) – digitoxin - a cardiotonic glycoside was found to enhance
cardiac conduction, thereby improving the
• Cinchona succirubra - drug quinine - anti-malarial fever, indigestion, mouth and throat
• diseases and cancer.
• Pilocarpus jaborandi (Rutaceae)- Pilocarpine (an L-histidine-derived alkaloid) - treatmentof
chronic open-angle glaucoma and acute angle-closure glaucoma for over 100 years. In
1994, an oral formulation of pilocarpine (6) was approved by the FDA to treat dry mouth
(xerostomia) which is a side effect of radiation therapy for head and neck cancers and also
used to stimulate sweat glands to measure the concentrations of sodium and chloride. In
1998, the oral preparation was approved for the management of Sjogren's syndrome, an
autoimmune disease that damages the salivary and lacrimal glands.
 Natural Products from Fungi

One of the most famous natural product discoveries derived from a fungus
(microorganism) is that of penicillin (7) from the fungus, Penicillium notatum
discovered by Fleming in 1929. A countercurrent extractive separation technique
which produced 7 in high yields was required for the in vivo experimentation that
ultimately saved countless lives and won Chain and Florey (together with
Fleming) the 1945 Nobel prize in Physiology and Medicine (Figure 2) [34]. This
discovery led to the re-isolation and clinical studies by Chain, Florey and co-
workers in the early 1940s and commercialization of synthetic penicillins, which
ultimately revolutionized drug discovery research[35–38].
 Natural Products from the MarineEnvironment

Though plants have proven to be a novel source for bioactive natural products the
marine environment has a clear track record in also offering novel structural
entities. “We are not marine organisms”, says Fenical, “so until about 1970, no one
even thought of the ocean. It was left as a deep secret. It seemed ridiculous to me
that the ocean — with such a vast habitat — had escaped anyone's notice. But
there are good reasons. People fear the ocean; it has been considered a very
hostile, inhospitable place”. Given that 70% of planet earth’s surface is covered by
ocean, pharmaceutical companies began to realize that the ocean would possess
unique biodiversity and may be a possible source for potential drug candidates.

Exploration of the marine environment and organisms (algae, sponges, ascidians,

tunicates and bryozoans) became possible due to modern snorkeling, the
introduction of SCUBA (1970s), to the use of manned submersibles (1980s) and
more recently the use of remotely operated vehicles (ROVs) (1990s).
 Natural Products as Sources of New Drugs
(Ref: Mark Butler, “The Role of Natural Product Chemistry in Drug
Discovery,” J. Nat. Prod. 2004, 67, 2141-2153)
 Some milestones in natural products chemistry:

H
HO
3 1
H N
4
N
HO
12 10
H
O 9
13
N H CH3O
5
H H
15 N 10
6 CH3 O
H O
7 16
N
HO

Morphine
(aromatic alkaloid from opium,
Papaver somniferum)
Isolation: 1806, Sertürner
Structure: 1925, Robinson
Synthesis: 1954, Ginsberg
Biogenesis: 1959, Leete
Strychnine
Quinine
(aromatic alkaloid from
(quinoline alkaloid from
Strychnos nux-vomica)
Cinchona species)
Isolation: 1818, Pelletier &
Caventou Isolation: 1820, Pelletier &
Structure: 1946, Robinson Caventou
Synthesis: 1944, Woodward
Synthesis: 1954, Woodward
2001, Eichberg
 Some milestones in natural products chemistry:

10
CH3
CH3 N
1 CO2CH3
2 O N
O
9 8 CH
65
7 H
CH3 CH3 O

4 Coniine
Cocaine
Camphor (aliphatic alkaloid from
(aliphatic alkaloid from
(monoterpene from hemlock, Conium
Erythroxylon coca)
Cinamomum camphora) maculatum)
Isolation: 1859, Niemann
Isolation: 1845, Bouchardat Isolation: 1886, Ladenburg
Synthesis: 1923: Willstätter
Structure: 1926, Koller
 Some milestones in natural products chemistry:

21
24
20 23 26
18
CH3 25 OH N(H)CH3
27
CH2OH 19 11 13 17 CH CH CH3
CH3
14
O b glucoside 1 9
10 8
3 5
6
HO
Ephedrin
Cholesterol (aromatic alkaloid from
Salicin (steroid from gallstones) Ephedra equisetrina and
(aromatic alcohol from
Isolation: 1909, Windaus E. sinica; "ma huang")
Salix species)
Structure: 1932, Wieland Structure and synthesis:
Structure and synthesis: Synthesis: 1964, Johnson 1920, Späth and Göring
1906, Irvine Biogenesis: 1966, Cornforth
Some milestones in natural products chemistry:
arabinose OH
glucose
OH
Ginsengoside Rs2 OH HO
HO OH O
(Panax ginseng) O
O
O
Isolation and structure: OH
1962, Shibata
glucose
HO
HO O
Brevetoxin-A
HO O
AcO
O
(red tide toxin from
HO O
Gymnodinium breve)
HO
OH Structure: 1986, Clardy
glucose-6-acetyl Synthesis: 1987, Nakanishi
OH

.
O O
CHO

O . . .
O
. O
O

. .
O

. . O

O O
O
 Some milestones in natural products chemistry:
.
AcO O OH . .
H3C CH3 CO2H
O Ph O HO O HN .
O
CH3
Ph NH O OCH3
CH3 H
OH H O
HO AcO
PhCO2 HO O OH

Taxol
(antitumor diterpene from Dynemicin A
Pacific yew, Taxus species) (antibiotic polyketide from
Micromonospora chersina)
Isolation: 1971, Wani et al.
Structure: 1971, Wani et al. Structure: 1989, Matsumoto and Clardy
Synthesis: 1991, Nicolau
Biosynthesis: 1992, Tokiwa et al.
 Some milestones in natural products chemistry:

OH
OH

O
H
H OH O
H
O
O O
O
OH
O H O
O OH
O
(+)-Absinthin O
OH

(dimeric diterpene from Artemisia (-)-Littoralisone

absinthium L., an anthelmintic)
(neurotrophic growth factor, iridoid
Isolation: 1953, Herout from Verbena littoralis L.)
Structure: NMR: 1980, Beauharie,
Isolation and structure: 2001, Li
X-ray: 1985, Karimov
Synthesis: 2005, Mangion
Synthesis: 2004, Zhang
 Techniques used in natural products chemistry
1800 1850 1900 1950 1975 2000

Type of research undertaken:

Isolation, characterization
Pharmacognosy & Pharmacology
Organic synthesis
Chromatography Spectroscopy
Biogenetic studies
Biochemistry; Enzymology
Molecular Biology
Metabolonomics

Techniques used:
basic physico-chemical measurements
TLC column chrom GC HPLC / Electrophoresis
X-ray UV-vis IR MS / NMR
Radioisotopes
Enzymes Computational methods
Tissue culture
Mol Bio / Biotech
Combinatorial chem
Modern directions in natural products chemistry:
• Genomics of bacteria and plants
• Novel and efficient synthetic methods
• Genetic engineering of bacteria and plants
• Enzyme synthesis
• Computational methods and modeling
• High efficiency chromatography
• Spectroscopic methods
• High-throughput screening
• Synergism
• Biotransformation
Issues and challenges in Natural Products today
1. Loss of biodiversity
2. Intellectual Property Rights
• Patent protection (pharma companies)
• Biopiracy (source countries)
3. Western drugs:
a. High cost of drug development
b. New drug leads and targets
4. Herbal products:
a. Regulation
b. Improvement in quality
c. Elucidation of mechanism of action
2009 Annual Review
 Sustainable Drugs and Global Health Care
(Ref: Geoffrey A. Cordell, Quim. Nova, Vol. 32, No. 5, 1356-1364, 2009)
Research areas in natural products today
1.Structural elucidation (speed of analysis, sample throughput complexity
of structures)
2. Metabolonomics
3. Synergy and biotransformation
4. Biosynthesis
5. Biological activity
a. Ecological
b. Pharmaceutical properties / drug discovery
c. Healthcare and cosmetic products
6. Molecular biology and Biotechnology
7. Quantitative natural products chemistry
Natural products chemistry is at the intersection of
many fields:
Ecological Biochemistry

Taxonomy
Pharmaceutical Science

Entomology
Biochemistry
Natural
Products Combinatorial
Chemistry Chemistry

Biotechnology
Chemical synthesis

Molecular Biology Microbiology

• How to differentiate
between Primary and
Secondary metabolite?

• Search in the internet the

success stories of Primary and
Secondary metabolite.

• Share your findings in the

next class.
 Classification of Natural Products
Secondary compounds are grouped into classes based on similar structures,
biosynthetic pathways, or the kinds of plants that make them. Natural
products are generally grouped into five main classes
1. Terpenoids and steroids, are a vast group of substances—more than 35,000 are
known—derived biosynthetically from isopentenyl diphosphate. Terpenoids
have an immense variety of apparently unrelated structures, while steroids have
a common tetracyclic carbon skeleton and are modified terpenoids that are
biosynthesized from the triterpene lanosterol.
2. Alkaloids, like terpenoids, are a large and diverse class of compounds, with more
than 12,000 examples known at present. They contain a basic amine group in
their structure and are derived biosynthetically from amino acids.
3. Fatty acid–derived substances and polyketides, of which more than 10,000 are
known, are biosynthesized from simple acyl precursors such as acetyl CoA,
propionyl CoA, and methylmalonyl CoA. Natural products derived from fatty
acids, such as the eicosanoid prostaglandin E1, generally have most of the
oxygen atoms removed, but polyketides, such as the antibiotic erythromycin A,
often have many oxygen substituents remaining.
4. Nonribosomal polypeptides are peptidelike compounds that are biosynthesized
from amino acids by a multifunctional enzyme complex without direct RNA
transcription. The penicillins are good examples for this class.
5. Enzyme cofactors don’t fit one of the other general categories of natural
products and are usually classed separately.
 Secondary metabolite classes
• Peptides
• Alkaloids
– Mostly derived from amino acids and usually heterocyclic,
potent pharmacological activities
• Polyketides and fatty acids
– A large group of mainly acetate(C2) derived natural products
– Similar to fatty acids
– Includes many of the antibiotics
• Shikimate metabolites
– Simple aromatic molecules, including Coumarins and Flavonoids
• Terpenoids
– Largest family of natural products, Includes the steroids
• Many occur as glycosides
• Some are of mixed origin
(Polyphenols/phenolics)
 • More than 10,000 known

• Biosynthesized from simple

acyl precursors such as
Fatty acid- acetyl CoA, propionyl CoA,
derived and methylmalonyl CoA
substances
• Natural products derived
and from fatty acids, such as
polyketides prostaglandin E1, generally
have most of the oxygen
atoms removed
Apiaceae - Polyacetylenes

Water Hemlock
 Terpenoids
• Terpenes are generally dimers and polymers of 5-
carbon (precursors) called isoprene unit (C5 H8).

• Terpenoids often evaporate from plants and

contribute to the haze we see on hot sunny
days.

• Give scent, flavors, colors, medicine

• Three plant hormones are derived from the

terpenoid pathway.

16
IMPORTANTTT
 Terpenoids

• Terpenes are found in all plants, but differ from

species to species and play a variety of
functional roles in the plant.

• The latex found in plants of the Euphorbiaceae is

poisonous and likely plays a defensive role.
 Taxol

• Taxol is a terpenoid
• "the best anti-cancer agent” by National Cancer
Institute
• Has remarkable activity against advanced ovarian
and breast cancer, and has been approved for
clinical use.
Where does taxol come from?

Taxol is found in the bark of the tree.

Problems:
• Pacific yew tree is very slow-
growing.
• Harvesting taxol from the
bark kills the tree
• To treat one cancer patient
requires 60 pounds of Yew
tree bark, the equivalent of
three, one hundred year old trees
Pacific Yew

Taxus brevifolia Nutt.

What can we do?
 Phenolics

• Compounds that contain a fully unsaturated

six carbon ring linked to an oxygen are
called phenolics.

• Derived from aromatic amino acids, such as

phenylalanine, tyrosin, and trytophan.

• Some examples: Coumarins: antimicrobial

agents, feeding deterrents, and germination
inhibitors.
The largest group:

• Common biosythetic
origin from
phenylalanine
• Aromatic
• Ionize in presence of
a base
• Used in spices
• Tannins are used in
tanning leather
The Plant Phenolic
Compounds
Phenylpropanoids
and flavour /
fragrance
 Alkaloids
• Alkaloids generally include alkaline substances that have
nitrogen as part of a ring structure. More than 6500
alkaloids are known and are the largest class of secondary
compounds. They are very common in certain plant
families, especially:
• Most are derived from a
few common amino acids
• Fabaceae – peas and beans
(i.e., tyrosine,
• Asteraceae - sunflowers tryptophan, ornithine or
• Papaveraceae - poppies argenine, and lysine)
• Solanaceae – nightshade, tomato • Compounds have
nitrogen as part of a ring
• Apocynaceae - dogbanes
structure.
• Asclepiadaceae - milkweeds • Indole alkaloids is the
• Rutaceae - citrus largest group in this
family, derived from
tryptophan
• Widely used as medicine
 Alkaloids

The alkaloids important group of compounds, many

of which have pharmaceutical value. Most have a
bitter taste and are toxic to animals. They are
usually very species specific.
Alkaloids are:
Secondary Metabolites
Alkali-Like compounds…Difficult to be defined…But,
Generally known as
“All Organic Nitrogenous Compounds With a Limited
Distribution in Nature”… “have Physiological Activity”
Not homogenous group of compounds!
Found in plants, microorganisms.

Extracted from seeds, fruits, leave, roots and barks

• Non-peptidic, non-nucleosidic nitrogen containing cpds
usually derived from an amino acid.
• Found in plants, insects, amphibians, fungi, sponges
etc.
• Bitter tasting, generally white solids (exception -
nicotine is a brown liquid).
• Alkaloids are “secondary metabolites”, they are not
involved in primary metabolism.
• Most studied group of natural products
• Many have heterocyclic rings as a part of their structure
• Many are basic (“alkaline”, due to an unshared pair on
N)
Discovery:
Narcotine first alkaloid discovery
Coniine first alkaloid to have its structure established
and synthesized
Paclitaxel revolution in alkaloid

Naming: (ends withine)

From plant generic name (Atropine)
From specific plant yielding it (Cocaine)
From physiological activity (emetine)

From discoverer
Chemistry:

Alkaloids may contain one or more nitrogen atoms, as 1o, 2o,

3o & 4o.
Most of them contain oxygen
Found as free or nitrogen oxides
Degree of basicity depends on the structure
Converted to their salts when treated with H+, while when
treated with OH, they give up their free amine.
Properties:

Sparingly soluble in water… salts are freely soluble in water.

Free alkaloids are soluble in ether, chloroform and non-polar
solvent…important for isolation and purification
Crystalline to amorphous or liquid when lack Oxygen
Have bitter taste
Form double salts with heavy metals reagents (I, Hg),
Wagners, Mayer, and Dragendroff reactions
Functions:
Provide Nonspecific Basic compounds (N)
•Source for their associated acids
•End products
•Part of some metabolic sequences
•Defense

Plants which accumulate alkaloids develop even

when deprived the alkaloid
Plants which do not produce alkaloid survive when
administered alkaloid
 MORPHINE - A TYPICAL ALKALOID

basic due to the contains nitrogen

unshared pair
..
Plant source. N CH3
Most alkaloids
are found in
plants. heterocyclic ring

MeO O OH

Found only in the Opium Poppy - papaver somniferum

….. not ubiquitous.
There are three main types of alkaloids:

colchicine
Terpenoids or
purines
HOW ARE ALKALOIDS CLASSIFIED ?

Common classification schemes use either:

• The heterocyclic ring systems found as a
part of the compound’s structure.
- in terms of their BIOLOGICAL activity,
- BIOSYNTHETIC pathway (the way they are produced in the plant).

• The plant or plant family where they originate*

* The majority of alkaloids (>90%) are found in plants -

therefore, we will speak mostly about plants and their
biochemistry.
 HETEROCYCLIC RING SYSTEMS

N N N N
H H H

pyrrolidine pyrrole piperidine pyridine

N N N
N
H H
quinoline isoquinoline indole dihydroindole
HETEROCYCLIC RING SYSTEMS (cont)

H
N
N N

quinolizidine pyrrolizidine tropane

N N
N
C C N
N N
H

benzylisoquinoline purine b-phenylethylamine

 Some Examples of Classification
BY RING TYPE

MeO O
OMe -
O P OH H3C CH3
NH OMe N
MeO O + CH3

N psilocybin
N
emetine H

O
H3C N
N
N N
O N
H CH3
CH3 CH3
N

nicotine caffeine
 Amino Acid Precursors

from
from from tryptophan
ornithine H3CO NH2 H3C ornithine
N N
CO2CH 3
N from
H3CO O Ph
CH3 tyrosine
N OCH 3 N
O
nicotine mescaline cocaine
O O
HO H
from HO2C CH 3
tyrosine N strychnine
from H
tryptophan N
O HO
N
CH 3 H3CO
HO NH
from
tryptophan N
morphine lysergic acid HO
N
H3C
N
from
lysine NH O
MeO2C OH
N
N O H O
H from MeO N
OH tryptophan CO2CH3
R
Lycopodine Histrionicotoxin
R= -CH3 vinblastine
R= -CHO vincristine
 Some Examples of Classification
BY PLANT FAMILY : “Amaryllis” Alkaloids
OH
HO
MeO belladine
O

MeO
N
O
N
MeO lycorine
H
The other three
are biochemically OH
derived from MeO galanthamine
H
belladine.
H
N CH3 O N CH3
OH
O
MeO
O
tazettine
O daffodils
narcissus
These alkaloids are found in Amaryllidaceae lillies
etc
THE PURPOSE OF ALKALOIDS IN PLANTS (?)
The spectacular pharmacological properties of many of the
alkaloids keeps asking about their purpose in plants.

Many ideas have been advanced:

Defense Mechanisms Insect Repellants Herbivore Attractants
Nitrogen Storage Growth Regulation Insect Attractants
Vestiges of Old Metabolic Experiments Anti-fungals
Metal ion transport (chelates) Competitive Herbicides

What seems most likely is that there are many reasons why plants
elaborate alkaloids, and in many cases the purpose of the alkaloid
may be unique to a given plant.
• Camptothecin is an indole alkaloid, derived
from tryptophan.
• Has anticancer and antiviral activity
Where does camptothecin come from?

• Happy tree grows in the

southern China.

• Camptothecin is found in
most of the tissues of a
Happy tree.

Camptotheca acuminata
Happy tree
Minor Secondary Metabolites

• Secondary compounds often occur in combination

with one or more sugars. These combination
molecules are known as glycosides. Usually the
sugar is a glucose, galactose. But some plants have
unique sugars.
 Minor Secondary Metabolites

• Mustard oil glycosides are nitrogen-sulfur containing compounds that

occur in cabbage, broccoli, horseradish, watercress and other
members of the mustard family (Brassicaceae). They give the group
its characteristic taste and odor.
• Cyanogenic glycosides occur in several families of plants, but are
especially common in roses (Rosaceae) and peas (Fabaceae). They
are sugar containing compounds that release cyanide gas when
hydrolyzed.
• Cardiac glycosides effect vertebrate heart rate. Especially common in
milkweeds Asclepiadaceae.
Mustard Oil
Cyanogenic Glycosides
 Cardiac Glycosides

Common Milkweed Purple Foxglove

A Biosynthetic Approach of
Natural Products
 Definitions
• Biosynthesis is the term for the in vivo
synthesis of metabolites / natural products.

• The formation of a chemical compound by a

living organism.
• The pathways for generally modifying and
synthesizing carbohydrates, proteins, fats, and
nucleic acids are found to be essentially the same in
all organisms, apart from minor variations.

• These processes are collectively described as

primary metabolism, with the compounds involved
in the pathways being termed primary metabolites.
 The building blocks
• The building blocks for secondary
metabolites are derived from primary
metabolism.

• The number of building blocks needed is

surprisingly few.
• The most important building blocks employed
in the biosynthesis of secondary metabolites
are derived from:
1. Acetyl coenzyme A (acetyl-CoA)
2. Shikimic acid
3. Mevalonic acid
4. 1-deoxyxylulose 5-phosphate
5. Amino acids
Secondary metabolites are derived
from primary metabolites

126
• Organisms vary widely in their capacity to synthesize
and transform chemicals.

• For instance, plants are very efficient at synthesizing

organic compounds via photosynthesis from
inorganic materials found in the environment, whilst
other organisms such as animals and microorganisms
rely on obtaining their raw materials in their diet,
e.g. by consuming plants.
 1. Acetate pathway
• The form in which acetate is used in most of its
important biochemical reactions is acetyl coenzyme
A (acetyl-CoA).
• Acetyl-CoA is formed by oxidative decarboxylation
of the glycolytic pathway product pyruvic acid.
• Important secondary metabolites formed from the
acetate pathway includes:
1. Phenols
2. Prostaglandins
3. Macrolide antibiotics
Coenzyme A: present in all living cells that
functions as an acyl group carrier.

NH2
N
N
O O H3C CH3 N
OH OH N
RS O O O
N N P P
H H O
OH O O
HO
HO O OH
P
O

Coenzyme A acts as an acyl transfer/α-

carbon activation reagent by forming
reactive acyl thioesters
 2. Shikimate pathway
• Shikimic acid is produced from a combination of
phosphoenolpyruvate, a glycolytic pathway
intermediate, and erythrose 4-phosphate from the
pentose phosphate pathway.
• The shikimate pathway leads to a variety of:
1.Phenols
2.Cinnamic acid derivatives
3.Lignans
4.Alkaloids
 3. Mevalonate pathway
• Mevalonic acid is itself formed from three
molecules of acetyl-CoA, but the
mevalonate pathway channels acetate into a
different series of compounds than does the
acetate pathway.
4. Deoxyxylulose phosphate pathway

• Deoxyxylulose phosphate arises from a

combination of two glycolytic pathway
intermediates, namely pyruvic acid and
glyceraldehyde 3-phosphate.
• The mevalonate and deoxyxylulose phosphate
pathways are together responsible for the
biosynthesis of a vast array of terpenoid and
steroid metabolites.
 5. Amino acids pathway
• Peptides, proteins, alkaloids and many antibiotics
are derived from amino acids.

• Intermediates from the glycolytic pathway and the

Krebs cycle are used in constructing many of them.

• The aromatic amino acids phenylalanine, tyrosine,

and tryptophan are themselves products from the
shikimate pathway.
• Secondary metabolites can be synthesized by combining
several building blocks of the same type, or by using a
mixture of different building blocks.
• Many of secondary metabolites also contain one or more
sugar units in their structure.
• To appreciate how a natural product is elaborated, it is of
value to be able:
1. To dissect its structure into the basic building
blocks from which it is made up.
2. To propose how these are mechanistically joined
together.
• Oxygen atoms can be introduced and removed
by a variety of processes, and so are not
considered in the initial analysis, except as a
pointer to an acetate or shikimate origin.

• Relatively few building blocks are routinely

employed, and the following list includes those
most frequently encountered in producing the
carbon and nitrogen skeleton of a natural
product.
 The construction mechanisms
• Natural product molecules are biosynthesized by a
sequence of reactions which are catalyzed by
enzymes.
• Most proteins in living cells are enzymes.
• Enzymes have the power to effect these
transformations:
1. More efficiently and more rapidly than the chemical
analogy.
2. Under very much milder conditions.
3. Carry out reactions in a stereospecific manner.
Pharmacological
Screening of
Drugs from
Natural Product
SAS
Drug Development Process
 Preclinical trial - a laboratory test of a new drug or a series of
chemicals, usually done on animal subjects, to see if the hoped-for
treatment really works and if it is safe to test on humans.
What is
preclinical
trial?
 Identify a drug Develop a
target bioassay

Several Steps in
a Pre-Clinical Screen the drug Establish effective
in assay and toxic doses
Trial

File for approval as

an Investigational
New Drug (IND)
Screening of drugs

A thorough investigation to:

A) Get pharmacological activity of new/chemically undefined substances

B) investigate the functions of endogenous mediators

C) measure / define the toxicity and or unwanted actions

 SCREENING: - A test or
group of test believed to
permit the detection of
physiological activity
Screening of
drugs

EVALUATION- - To reduce
the uncertainty of scanning
 Is the substance active?
Simple (1/2 similar testing)

Is there any
Biological activity ?

Blind

Programmed

In what ways compound(s) acts ?

Main activity ?.,,,,, Subsidiary activity?
7
 Only for the series of new chemical substances with no prior
pharmacological history.
New chemical entity or isolated naturals

Provides a road towards fields of activity if they exists

To demonstrate whether new group of substances is worthy

BLIND for further attention?
SCREENING Point out the most potent chemical with interesting
pharmacological activity
Requires planning and skillful execution of test (Nature,

economical, time & money)

Toxicological pathway is essential for every library of

compounds
 Provide information “what compounds are active in what ways?”

A new drug of specific type (known)and/or series of

chemicals is to be investigated for some particular pharmacological activity
(e.g. System or organ specific)

A series of testing program is required to provide information on the

compounds on specific targets
PROGRAMMED
SCREENING
Explores main activity and subsidiary activity

Potency can be compared with known compounds which lead the

investigator to proceed or to terminate

More limited than blind, precision is expected

 Preclinical Studies

Invitro Invivo

Ø Receptor Characterization
Pharmacological Toxicological
Ø Receptor binding assay Studies studies
Ø Enzyme inhibition
Ø 20 Messenger analysis Efficacy Safety
Ø Cytotoxic activity

Dose conversion

Determination of
starting dose

11
 12

METHODS OF SCREENING
• Invitro :
▫ Experimental process in a given procedure which is mainly
done outside the body in a controlled condition
Activity assays (screen the activity)
Bioassays (define the molecular mechanism)
Toxicity assays (Toxicity of chemicals)
Types: Biological assay using isolated tissues/organs
(skeletal/smooth muscles, aorta, heart etc.,)
Chemical Assay using regents Antioxidant assays
Cell culture studies Xanthine oxidase activity
Antiglycation activity
Toxicity(cyto) assays
DNA, protein, RNAlevel
Immunological assays
assays
Cancer cell line studies
Immunological assays
 13

• Exvivo:
▫ Experimental process which is performed outside the living
body in an ‘artificial invivo environment’
▫ This usually lasting up to 24 hrs
• In vivo
▫ Experimental process which is performed in the living body
using laboratory animals

• Insilico
▫ Process which is performed on computer or via computer
simulator
 Techniques employed for the determination of the potency of
chemical and biological agents like drugs, hormones, ions, etc.,
by means of biological indicators using whole animals, isolated
organs and tissues or using cell lines

Biological Indicators:

ü Body temperature
ü Blood glucose level
ü Behavioral responses
ü Serum parameters
ü Contraction/relaxation
ü Growth/inhibition of cells
13
 Factors:
Species
STEP I Strains
◦ Toxicological assessment of chemicals Sex
◦ LD50 estimation Age
Disease
Using Acute/sub-acute, chronic etc., studies Induction
STEP II ( Environmental

◦ Evaluation of 10 & 20Pharmacological activity

◦ Animal models of induced disease and injury
STEP III
◦ ADME Studies
Absorption studies
Tissue/organ/fluid conc. estimation
Histopathological studies
Serum estimation of biological indicators for drug & metabolites

14
Studies to be performed before
clinical trials
Single dose toxicity in Repeated dose toxicity in
Pharmacodynamics Pharmacokinetics
two species two species,

Chronic
Safety Pharmacology Local tolerance Genotoxicity
Toxicity/Carcinogenicity

Reproductive Toxicity/
Teratogenicity study

15
Principles of
Toxicology:
The Study of Poisons
Adverse effects
any change from an
organism’s normal state
dependent upon the
The study
of the
concentration of active
compound at the target site
for a sufficient time.

adverse
effects of a
any agent capable of

Toxicant (Poison) producing a deleterious

response in a biological
system

toxicant on
Living organism
a sac of water with target
sites, storage depots and
living
enzymes
organisms
 • Phillip von Hohenheim (1493 -
1541),(Paracelcius) was an alchemist,
physician,astrologer and known as the“
History father of toxicology“ “All things are poison
and nothing is without poison, only the
dose permits something not to be
poisonous.”
 All substances are poisons;

there is none that is not a poison.

What is a The right dose
Poison?
differentiates a poison and a remedy.

Paracelsus (1493-1541)
 Toxin
• Toxic substances that are produced naturally (nature origin)

Toxic
• This term relates to poisonous or deadly effects on the body

Definition Of Toxicants
Terms • Any chemical that can injure or kill humans, animals, or
plants; a poison
Toxicity
• Describes the degree to which a substance is poisonous or
can cause injury. The toxicity depends on a variety of
factors: dose, duration and route of exposure, shape and
structure of the chemical itself, and individual human
factors.
 The amount of mg of chemical/kg
This is usually
chemical entering of body weight =
given as
the body mg/kg

Dose The dose is

dependent upon
The environmental
concentration
The properties of
the toxicant

The frequency of The length of The exposure

exposure exposure pathway
What is a Response?
The degree and spectra of responses depend upon the dose and the
organism--describe exposure conditions with description of dose

Change from normal state

◦ could be on the molecular, cellular, organ, or organism level--the
symptoms

Local vs. Systemic

Reversible vs. Irreversible
Immediate vs. Delayed
Graded vs. Quantal
◦ degrees of the same damage vs. all or none
 Dose-Response Relationship:
As the dose of a toxicant increases,
so does the response.
4

RESPONSE

0-1 NOAEL
2-3 Linear Range 3
4 Maximum Response

0 1 DOSE
DOSE DETERMINES THE BIOLOGICAL RESPONSE
 Quantal responses can be treated as
gradient when data from a population is
used.
The cumulative proportion of the population
responding to a certain dose is plotted per
dose--10-30 fold variation w/in a population
LD50
If Mortality is the response, the dose that is
lethal to 50% of the population LD50 can be
generated from the curve
Different toxicants can be compared--lowest
dose is most potent
LD50 Comparison
Chemical LD50 (mg/kg)
Ethyl Alcohol 10,000
Sodium Chloride 4,000
Ferrous Sulfate 1,500
Morphine Sulfate 900
Strychnine Sulfate 150
Nicotine 1
Black Widow 0.55
Curare 0.50
Rattle Snake 0.24
Dioxin (TCDD) 0.001
Botulinum toxin 0.0001
 Routes and Sites of Exposure
Injection
Ingestion
Inhalation Dermal/Topical • intravenous,
(Gastrointestinal
(Lungs) (Skin) intramuscular,
Tract) intraperitoneal

Exposure:
Pathways
Typical Effectiveness of Route of Exposure

iv > inhale > ip > im > ingest > topical

 •Skin
Routes of •Chemicals that can
Exposure penetrate healthy intact
skin – aniline, hydrogen
cyanide,
organophosphate, etc.
•Absorption through skin from
the chemical that absorbed
through clothing is far more
worse
Routes of
Exposure
•Lung (Inhalation)
•Depends on
•Size & Shape of particles
•Rate of physical work (Tidal
Volume increase by
exertion)
Routes of • Size & Shape of particles
Exposure • Size – effective aerodynamic diameter
• Shape – dust, microorganism
(Lung) • Larger diameter (>10 micro meter)
lodge in bronchi/bronchioles
mucocilliary clearance oesophagus
Gut
• Smaller Diameter(<2 micro meter)
persist in alveoli cause harm
• e.g. Insoluble particle (Asbestos)
macrophage tried to engulf but
damaged hydrolytic enzyme leak
local tissue damage fibrosis
Routes of Exposure
(Lung)
• Rate of physical work
• Advice to avoid physical activity
during haze
Routes of
Exposure
• Ingestion
• Mostly we can control (unlike airborne)
• Airborne particle also can be ingested
• Depends on
• Concentration
• Time
• Continuous
• Intermittent
• Sometimes can accumulate and cause harm
in later life e.g. Lead which accumulates in
bones cause little harm but once broken, can
cause harm to the body
Adverse
effect
• Characteristics
• Local
• Systemic
• Both Local & Systemic e.g. Allergic reaction
• Accumulation
• Chemical e.g. Adipose tissue accumulate organochloride pesticide and does not
cause harm
• Damage e.g. death of nerve cell following repetitive exposure
• Factors
• Balance between Absorption and excretion
• Balance between injury and repair
• Immediate or delayed effect
• Reversible or irreversible
 Adverse effect

• Local
• Irritants
• Corrosive
• Systemic e.g. Organophosphate
poisoning
Chemical
Interaction
• Additive effects ( 1 + 1 = 2)
• Synergistic Effects ( 1 + 1 = 4)
• Antagonist (1 + 5 = 2)
 Decrease in sensitivity
to a chemical following
exposure
Tolerance and
resistance
Resistance à complete
insensitivity towards
chemical
 Acute < 24hr usually 1 exposure
Subacute 1 month repeated doses

Subchronic 1-3mo repeated doses

Exposure: Chronic > 3mo repeated doses
Duration
Over time, the amount of chemical in the body can
build up, it can redistribute, or it can overwhelm
repair and removal mechanisms
Classification
Classification of toxic
agents
• Toxic substances are classified into the following
1. Heavy Metals
2. Solvents and Vapours
3. Radiation and Radioactive Materials
4. Dioxin/Furans
5. Pesticides
6. Plant Toxins
7. Animal Toxins
Effect of toxic agents (Heavy metal)

• Arsenic
• Inorganic arsenic is a known carcinogen and can cause cancer of the skin, lungs, liver and
bladder
• Barium
• Barium is not known to cause cancer
• Short term exposure can cause vomiting, abdominal cramps, diarrhoea, difficulties in breathing,
increased or decreased blood pressure, numbness around the face, and muscle weakness
• Large amounts of barium intake can cause, high blood pressure, changes in heart rhythm or
paralysis and possibly death.
 • Cadmium
• Cadmium and cadmium compounds are
known human carcinogens
• Smokers get exposed to significantly higher
cadmium levels than non-smokers
• Severe damage to the lungs may
Effect of toxic occur through breathing high levels
of cadmium
agents (Heavy • Lead
• Exposure to high lead levels can severely
metal) damage the brain and kidneys and
ultimately cause death
• In pregnant women, high levels of exposure
to lead may cause miscarriage
• High level exposure in men can
damage the organs responsible for
sperm production.
 Mercury
• Exposure to high levels can permanently damage the brain,
kidneys, and developing foetuses
• Effects on brain functioning may result in irritability, shyness,
tremors,
• changes in vision or hearing, and memory problems
Effect of toxic Selenium
agents (Heavy • Chronic oral exposure to high concentrations can produce
selenosis
metal) • Major signs of selenosis are hair loss, nail brittleness, and
neurological
• abnormalities
• Brief exposures to high levels in air can result in respiratory
tract irritation, bronchitis, difficulty breathing, and stomach
pains
• Longer-term exposure can cause respiratory irritation,
bronchial spasms, and coughing.
 • Benzene
• Benzene enters the body through
inhalation and it may pass through the
skin
• Exposure to low concentrations
Effect of toxic may
cause
agents (Solvent dizziness, lightheadedness,
and vapours) headache, loss of appetite and
stomach upset
• High exposures to benzene may cause
irregularities in the heart beat which
can lead to death
• It has carcinogenic effect as well.
 • carcinogens (e.g., benzene, carbon
tetrachloride, trichloroethylene)
Effect of toxic • reproductive hazards (e.g., 2-
agents (Solvent ethoxyethanol, 2-methoxyethanol,
methyl chloride)
and vapours)
• neurotoxins (e.g., n-hexane,
tetrachloroethylene, toluene)
Effect of toxic agents
(Radiation and radioactive
material)
• Short-Term Health Effects of Radiation Exposure and Contamination
• Acute Radiation Syndrome (ARS)à a serious illness that can happen when a
person is exposed to very high levels of radiation, usually over a short period
of time.
• Symptoms of ARS may include nausea, vomiting, headache, and diarrhea
• Long-Term Health Effects of Radiation Exposure and Contamination
• Cancer
• Prenatal radiation exposure
• Mental health
 Short-term exposure of humans to high
levels of dioxins may result in skin lesions,
such as chloracne and patchy darkening
of the skin, and altered liver function
Effect of toxic
agents (Dioxin/
furans)
Long-term exposure is linked to
impairment of the immune system, the
developing nervous system, the endocrine
system and reproductive functions.
Effect of toxic agents (Pesticides)
• Organochlorines à cause a loss of sensation around the mouth,
hypersensitivity to light, sound, and touch, dizziness, tremors, nausea,
vomiting, nervousness, and confusion
• Organophosphates and Carbamates à causes signs and symptoms
of excess acetylcholine, such as increased salivation and perspiration,
narrowing of the pupils, nausea, diarrhea, decrease in blood pressure,
muscle weakness, and fatigue
• Pyrethroids à Pyrethroids can cause an allergic skin response, and
some pyrethroids may cause cancer, reproductive or developmental
effects, or endocrine system effects
Effect of toxic agents (Plant toxins)
Plants produce a range of chemicals designed to fend off predators or
discourage consumption by insects or animals.
• Philodendron, poison ivy, cashew à allergic dermatitis
• Grassesà allergic rhinitis
• Lily family, glory lily, crocus, horse chestnut à affects the GIT tract
Effect of toxic agents (Plant toxins)
• Red alga (red tide), green alga, mushrooms, Coffee bean, tea, cola
nut mint family à affects the nervous system
• Fungus that grows on peanuts, walnuts à liver cancer
• Legumes (Astrogalus); bitter melon seeds (Momordica) à affects
the reproductive system
Effect of toxic agents (Animal toxins)
These toxins can result from venomous or poisonous animal releases
• For examples
• scorpions, spiders , ticks à produces neurotoxin
• Rattlesnakes, cobras, coral snakes à produces very complex enzyme-based
venoms and neurotoxin
Toxicokinetic &
Toxicodynamic
Toxicodynamic
• Toxic ( The Chemical) + Dynamic
(Changes, Perubahan)
• Toxic action on living system
• E.g. excessive ethanol injure liver
by blocking metabolism of fat &
carbohydrate, and scar tissue
replace healthy tissue causing
Liver cirrhosisà this process is
toxicodynamic
Toxicodynamic

• Dose-Toxicity relationship
• Dose-effect relationship
• Biological effect monitoring
• Dose –Response relationship
• Acute & Chronic effects
• Toxicity testing & health risk
 Dose-Response and
Concentration response
relationship

Toxicity Testing
Fixed dose testing

• Toxic
• Very Toxic
• Harmful
Dose-Response
relationship
• Incidence of defined biological effect in an
exposed population, expressed by
percentage
• LDn = Dose of toxicants lethal to n % of
population
• LD50 = Single dose of chemical that can cause
death in 50% of population in an
experimental condition e.g. death of mice
• LD50 does no tell sub lethal toxicity & does
not explain shape of dose-response curve
that it derive (Figure 1.2)
• Threshold dose à minimal dose required for
detectable response, expressed as
NOEL/LOEL (No/Lowest Observed Effect
Level)
Toxicokinetic
• Toxico + Kinetics (Pergerakan)=
movement of chemicals around
the body
• E.g. Ethanol from Beerà
Acetaldehydeà Acetic Acid à
nasty odour, used in
breathalyser = This is
Toxicokinetic (The way body
handle potentially toxic
substance)
Toxicokinetic
• The study of
• Absorption
• Distribution
• Metabolism
• Excretion
ADME:
Absorption, Distribution, Metabolism, and Excretion

Once a living organism has been exposed to a toxicant, the compound must get into the body and to
its target site in an active form in order to cause an adverse effect.

The body has defenses:

◦ Membrane barriers
◦ passive and facilitated diffusion, active transport

◦ Biotransformation enzymes, antioxidants

◦ Elimination mechanisms
 Inhalation--readily absorb gases into the
blood stream via the alveoli. (Large alveolar
Absorption: surface, high blood flow, and proximity of
ability of a blood to alveolar air)

chemical to Ingestion--absorption through GI tract

stomach (acids), small intestine (long
enter the blood contact time, large surface area--villi; bases
(blood is in and transporters for others)
◦ 1st Pass Effect (liver can modify)
equilibrium
Dermal--absorption through epidermis
with tissues) (stratum corneum), then dermis; site and
condition of skin
Distribution: Blood carries the agent to and from its site of

the process in action, storage depots, organs of

transformation, and organs of elimination
which a Rate of distribution (rapid) dependent upon
chemical agent ◦ blood flow
translocates ◦ characteristics of toxicant (affinity for the
throughout the tissue, and the partition coefficient)

body Distribution may change over time

 • Storage in Adipose tissue--Very lipophylic
compounds (DDT) will store in fat. Rapid
mobilization of the fat (starvation) can
Distribution: rapidly increase blood concentration
• Storage in Bone--Chemicals analogous to
Storage and Calcium--Fluoride, Lead, Strontium
Binding • Binding to Plasma proteins--can displace
endogenous compounds. Only free is
available for adverse effects or excretion
 Not all organs are affected equally
◦ greater susceptibility of the target organ
Target Organs: ◦ higher concentration of active compound
adverse effect is Liver--high blood flow, oxidative reactions
dependent upon Kidney--high blood flow, concentrates chemicals
the Lung--high blood flow, site of exposure
concentration of Neurons--oxygen dependent, irreversible
active compound damage
at the target site Myocardium--oxygen dependent
for enough time Bone marrow, intestinal mucosa--rapid divide
Adverse effects can occur at the
level of the molecule, cell, organ, or
organism

Molecularly,
Target Sites:
chemical can
interact with
Proteins Lipids DNA
Mechanisms
interfere with receptor-ligand
of Action
binding
interfere with membrane
Cellularly, chemical function

can interfere with cellular energy

production
bind to biomolecules
perturb homeostasis (Ca)
 Urinary excretion
◦ water soluble products are filtered out of the
blood by the kidney and excreted into the
Excretion: urine

Toxicants are Exhalation

eliminated from ◦ Volatile compounds are exhaled by breathing

the body by Biliary Excretion via Fecal Excretion

◦ Compounds can be extracted by the liver and
several routes excreted into the bile. The bile drains into the
small intestine and is eliminated in the feces.

Milk Sweat Saliva

The process by which the administered
chemical (parent compounds) are modified Metabolism:
by the organism by enzymatic reactions.
adverse effect
depends on the
1o objective--make
concentration of
decrease lipid solubility
--> decrease amount at
chemical agents more target
water soluble and easier
to excrete
increase ionization
--> increase excretion rate -
-> decrease toxicity
active
compound at
the target site
Bioactivation--Biotransformation can result in
the formation of reactive metabolites over time
Biotransformation (Metabolism)
Can drastically effect
the rate of clearance
Compound Without With
of compounds
Metabolism Metabolism
Ethanol 4 weeks 10mL/hr

Can occur at any Phenobarbital 5 months 8hrs

point during the
compound’s journey DDT infinity Days to weeks
from absorption to
excretion
 Key organs in biotransformation

• LIVER (high)
• Lung, Kidney, Intestine (medium)
• Others (low)
Biotransformation
Biotransformation Pathways

• Phase I--make the toxicant more water

soluble
• Phase II--Links with a soluble
endogenous agent (conjugation)
 Genetics-species, strain variation, interindividual
variations (yet still can extrapolate between
Individual mammals--similar biological mechanisms)
Susceptibility
Gender (gasoline nephrotox in male mice only)
--there can be 10-30
fold difference in Age--young (old too)
◦ underdeveloped excretory mechanisms
response to a toxicant ◦ underdeveloped biotransformation enzymes
in a population ◦ underdeveloped blood-brain barrier
 Age--old
• changes in excretion and metabolism
rates, body fat
Nutritional status
Individual
Susceptibility Health conditions

Previous or Concurrent Exposures

• additive --antagonistic
• synergistic
 Exposure + Hazard = Risk

All substances can be a poison

Dose determines the response

Pathway, Duration of Frequency of Exposure and Chemical determine Dose

Toxicology
Absorption, Distribution, Metabolism & Excretion

The extent of the effect is dependent upon the concentration of the active
compound at its site of action over time

Bioactivation: compounds to reactive metabolites

Individual variation of the organism will affect ADME

Risk
Asessment
 Toxicologic risk assessment
• Population at risk.
1. Hazard • Adverse health effects.
identification

• Epidemiological and experimental dose-response data.

• “critical” dose-response relationship.
2. Dose-response
• Quantitative expression of the dose-response relationship.
assessment

• Past, present and future exposure levels.

3. Exposure
assessment

• Estimate incidence of adverse health effects, in the pop predicted from the dose-response
4. Risk assessment (Step 2) as applied to the exposure assessment (Step 3).
characterization

46
Management
• At presentation
• History from all reliable source
(patients, family,co-workers)
• Physical examination
• Lab investigation for suspected toxin
HistoryTaking
• History is the most valuable tool.
• In some patient (comatose, drowsy, atered conciousness), family,
friends, relative,1st medical personel on scene should be questioned.
• All suspected possible toxins should not be missed
• When possible, patient’s house and workplace should be examined
(not only for toxins but also other things like recrational drugs, empty
medicine container or suicide note)
• If in doubt, extra information can be
obtained from
` • Poison Control Centres (Pusat
Racun Negara)
• Material safety Data Sheet (
available in
almost industrial plant)
 • Starts with the examination of vital signs ( GCs, BP,
HR etc).
• Then check for signs suggesting of toxicity
• Ingested/absorbed toxin –look for systemic
manifestation
• Corrosive toxin –check for GI tract
• Skin contact- acute cutaneus syndrome
Physical (blister,rashes, pain)
• Inhalated toxin
• Water soluble ( eg ammonia ,Chlorine) –upper
examination airways symptoms
• Less water soluble (phosgene)- look for lower
airway symptoms
• In some cases of toxicity (especially chronic
esposure to toxin), the altered
conciousness can be due to other causes
like hepatic enchelopathy, Wernicke
encelopathy and hypoglycemia.
Laboratory testing

Currently, lack of Measurement of

Can be standard readily
toxin blood level
qualitative or available test to
can help in
quantitative identify all the
toxins maanagement
 Management (not sure if all done in
Malaysia setting)
• Stabilization
• Maintain airway, breathing, circulation
• Pt without pulse, BP requird resuscitation
• Mechanical ventilation might be needed depending on cases
• IV fluids
• Topical Decontamination
• Any body surface (plus eyes) that are exposed to toxin is flushed with large
amount of saline or water
• Contaminated clothing, ewellery, accessories should be removed
• Acticated charcoal (in suspected oral toxicity)
• Should be given as early as possible
• Has not been proven to reduce mortality/morbididy

• Chelating agent-in metal toxicicty cases

Chelaating Drug Metal
Deferoxamine Iron
Dimercaprol Antimony
Arsenic Bismuth
mercury

Edate Ca disodium Cobalt, lead, zinc

Penicillamine Arsenic, cooper,Lead
Succimer Arsenic
Dialysis

• common in ethylene glycol, lithium,methanol,and salicylates poisoning

• Less effective if
• Toxin is large/charged molecule
• Bound strongly to protein
• Has large volume of distribution

Intentional use of toxin need psychiatric evaluation

Examples

58
Farmers and Farm Personnel

Potential exposures to toxicants resulting from:

• Fertilizer use
• Equipment use
• The use of pesticides and fumigants
• Animal confinement facilities
• Silo

59
Exposures Descriptions
Anhydrous • Odor warning threshold = 53ppm = provide margin of safety.
ammonia • 400 ppm = irritation of eyes, nose, throat.
fertilizer • 700ppm = immediate eye injury..
• 2500 to 4500 ppm for 30 minutes = lethal.
• 5000 pm = rapidly fatal.
• Upper airway edema à cyanosis and asphyxiation
• Chr sequelae = bronchiolitis obliterans and chr cystic bronchiectasis.
Farm equipment • Oral siphoning of gasoline with a rubber hose à ingestion and aspiration.
• Aspiration of hydrocarbon incl gasoline à severe lung injury,
• Welding hazards à inhalation of metal fumes, ozone, NO2, CO2.

Animal • Oxygen depletion near surface of the manure.

confinement • Direct exposure to methane and CO2.
• Respi problems = organic toxic dust syndrome, acute and chr bronchitis, occupational
asthma, COPD, hypersensitivity pneumonitis.
Toxicity of • Very large exposures from ingestion, dermal contact, and inhalation.
pesticides • Large oral exposures à N&V, diarrhea, pulm edema, cardiac arrhythmias.
• Dermal exposure à chemical burns (painful parasthesias, m stiffness).

60
Doctors, nurses and dentists
Potential toxic Pathophysiology
exposures
Mercury • Mercury combines easily with metals eg gold, silver, and tin to form alloys
called amalgams à used in dental fillings.
• Exposure to mercury vapor occurs from instruments that mix amalgam
(mechanical amalgamators), sterilizing instruments contaminated with
amalgam, and handling, storing, or cleaning mercury or amalgam.
• Elemental mercury used in Cantor tubes, thermometers, and
sphygmomanometers.
• Acute elemental mercury inhalation = local pulmonary toxicity.
• Low-level chr exposure = CNS effects eg weakness, fatigue, anorexia, GI
disturbances.
• Blood or urine mercury levels.

Waste anaesthetic • Leaking gas delivery systems, scavenger system.

gases • Route = inhalation.
• Toxic effects: Nitrous oxide peripheral neuropathy, halothane, hepatitis.

61
As a scientist
List down the risk that you might encountered in lab for your FYP

How you overcome it

Methods of
Extraction and
Separation
 Microbes:
Isolation, identification and
culturing of
Plants/animals/aquatic
organisms:
• Identification /and
conservation (in situ/ex
situ)
• Ethnobotany vs.
random sampling

Collection, Storage, and Vouchering of Plants

• Collection of Plants in the Field: Do’s and Don’ts
• Storage of Plants at Low Temperatures
• Vouchering of Plants Collected in the Field
1. Keep a Good Logbook
2. Photographic Records of Plants Collected
3. Preparation of Dry Specimens
4. Living Plant Specimens
A. Collection,
Storage, and
Vouchering of
Plants
• If you are collecting live plants on a longer trip, or you do not have access to liquid nitrogen, take
some Ziploc® plastic bags of various sizes in which to put the samples after collecting plants on site.
Such bags keep the plant materials alive until they can be frozen. Slips of recycled waterproof paper
or flagging tape are good to have so you can place notes on plant identity with your collected
specimens that match up with your field notes about the respective collections.
• It is important to get representative samples of all parts available: roots, vegetative shoots,
bark from stems (if woody plant), flowers, fruits, and seeds (if mature).
• When collecting plants in the field, do not take every last plant in the population,
especially if the plant is rare, threatened, or endangered.
• In the process of collecting herbaceous perennial plants (plants that come from the same
mother plant year after year), leave some of the original plant intact where it is growing so that it
can reproduce during the current and following years. Many of these plants take years to produce
even a small amount of new biomass every year.
• If you are collecting mushrooms or puffballs in the field, wrap the fruiting bodies in wax paper and
place them in a collecting basket or other suitable container where they will not become
squashed. This will help for later identification and for making spore prints from the fruiting
bodies.
• Do, thank the plant for providing you with substrate for your extracts. While this may not
seem important to some, we all do this in various ways when we collect plants in the home garden
for food or for aesthetic purposes, or when we collect wild edible plants in the field.
 2. Storage of Plants at Low
Temperatures
• The best way to do this is by quickly freezing the tissue in liquid
nitrogen. Such samples can then be stored long term in a –20 °C
or –80 °C commercial freezer. This technique prevents almost all
degradation of the plant material or any enzymatic changes that
alter or degrade naturally occurring metabolites. Such techniques
are especially important for molecular biological studies, because
once a plant is damaged by being picked, it undergoes a drastic
defense response that can greatly alter the composition of plant
compounds, such as RNA and proteins.

• Natural drying of the plant material can also be done if yield of

metabolites is not critical or dried at controlled temperature (40
°C)
 3. Vouchering of Plants Collected in the Field

• 3.1. Keep a Good Logbook

• Always keep a logbook of the plants and the tissues that were collected and
frozen.
• [collecting site location, soil conditions, ecological habitat, environmental
conditions, as well as the date of collection, plant identity, and who collected
the plant]
• The environmental conditions help control the biosynthesis of various
compounds; so, a record of the collection conditions may help to avoid
confusion when comparing multiple collections of the same type of plant.
• For similar reasons, we recommend taking soil samples and later recording
information on soil nutrients, soil pH, and soil type where each plant grows. It is
also important that proper labels be kept on samples stored in the freezer. Slips
of paper work well inside Ziploc plastic bags at –20 °C.
3.2. Photographic Records of Plants Collected
It is often desirable to keep photographic records of plants that were collected, either
for plant identification or for re-collection of a particular plant. We recommend taking
digital photographs of the live plant in the field in addition to performing high-
resolution scans (using a flatbed computer scanner) back in the laboratory.
3.3. Preparation of Dry Specimens
Dried plant specimens are prepared in order to have them available at any time as voucher
specimens representing typical plants that were collected in the field and used for plant
extracts. They are also called herbarium specimens. Dried plant specimens are prepared in
the traditional way by placing the collected plant between single newspaper sheets and
then placing this in a sandwich consisting of a dry blotter above and below the newspaper
sheets. A piece of corrugated cardboard (with air spaces present) is then placed above and
below each blotter. Successive sandwiches are placed atop one another and then
compressed between two wood-slatted frames and tied together tightly with straps. The
entire assembly is then placed upright on its side over a heat source, such as a radiator or
a plant drier, with the heat on a moderate temperature (e.g., 35 to 40 °C). The specimens
are allowed to dry this way for 48 h or longer. If plant specimens are very high in water
content, it is a good idea to replace the blotters with dry ones in the middle of the drying
process.
3.4. Living Plant Specimens
In our experience, we found it to be a good idea to collect seeds and living
specimens of the plants that are to be used for the extraction of natural
products. We do this in order to have the living plants on hand (e.g., medicinal
plants, dye plants, or culinary herb garden or in a greenhouse) to be used for
later extractions or experimental treatments to enhance metabolite
biosynthesis; this is especially important when access to the original collecting
site is not possible or convenient.

In situ
Ex situ germplasm
In vitro
 Extraction
• Extraction is the first step to separate the desired natural products from the raw materials.
• Solvent extraction is the most widely used method. The extraction of natural products progresses
through the following stages: (1) the solvent penetrates into the solid matrix; (2) the solute dissolves
in the solvents; (3) the solute is diffused out of the solid matrix; (4) the extracted solutes are collected.
Any factor enhancing the diffusivity and solubility in the above steps will facilitate the extraction. The
properties of the extraction solvent, the particle size of the raw materials, the solvent-to-solid ration,
the extraction temperature and the extraction duration will affect the extraction efficiency
• Generally, the finer the particle size is, the better result the extraction achieves.
• High temperatures increase the solubility and diffusion. Temperatures that too high, however, may
cause solvents to be lost, leading to extracts of undesirable impurities and the decomposition of
thermolabile components.
• The extraction efficiency increases with the increase in extraction duration in a certain time range.
Increasing time will not affect the extraction after the equilibrium of the solute is reached inside and
outside the solid material.
• The greater the solvent-to-solid ratio is, the higher the extraction yield is; however, a solvent-to-solid
ratio that is too high will cause excessive extraction solvent and requires a long time for concentration.
Maceration

• This is a very simple extraction

method with the disadvantage of
long extraction time and low
extraction efficiency. It could be
used for the extraction of
thermolabile components.
Percolation

• Percolation is more efficient than

maceration because it is a
continuous process in which the
saturated solvent is constantly being
replaced by fresh solvent.
Decoction
• The extract from decoction
contains a large amount of
water-soluble impurities.
Decoction cannot be used
for the extraction of
thermolabile or volatile
components.
•
Reflux
Extraction
• Reflux extraction is more
efficient than percolation or
maceration and requires less
extraction time and solvent. It
cannot be used for the
extraction of thermolabile
natural products.
•
Soxhlet
Extraction
• The Soxhlet extraction method
integrates the advantages of the reflux
extraction and percolation, which utilizes
the principle of reflux and siphoning to
continuously extract the herb with fresh
solvent. The Soxhlet extraction is an
automatic continuous extraction method
with high extraction efficiency that
requires less time and solvent
consumption than maceration or
percolation. The high temperature and
long extraction time in the Soxhlet
extraction will increase the possibilities of
thermal degradation.
Pressurized Liquid
Extraction (PLE)
• Pressurized liquid extraction
(PLE) has also been described as
accelerated solvent extraction,
enhanced solvent extraction,
pressurized fluid extraction,
accelerated fluid extraction, and
high pressure solvent extraction by
different research groups. PLE
applies high pressure in extraction.
High pressure keeps solvents in a
liquid state above their boiling
point resulting in a high solubility
and high diffusion rate of lipid
solutes in the solvent, and a high
penetration of the solvent in the
matrix. PLE dramatically decreased
the consumption of extraction time
and solvent and had better
repeatability compared to other
methods.
Supercritical fluid
Extraction
• Supercritical fluid extraction (SFE) uses
supercritical fluid (SF) as the extraction solvent.
SF has similar solubility to liquid and similar
diffusivity to gas, and can dissolve a wide variety
of natural products. Their solvating properties
dramatically changed near their critical points
due to small pressure and temperature changes.
Supercritical carbon dioxide (S-CO2) was widely
used in SFE because of its attractive merits such
as low critical temperature (31 °C), selectivity,
inertness, low cost, non-toxicity, and capability to
extract thermally labile compounds. The low
polarity of S-CO2 makes it ideal for the extraction
of non-polar natural products such as lipid and
volatile oil. A modifier may be added to S-CO2 to
enhance its solvating properties significantly.
Ultrasound assisted
extraction (UAE)

• Ultrasound assisted extraction (UAE)

• Ultrasonic-assisted extraction (UAE), also called
ultrasonic extraction or sonication, uses ultrasonic
wave energy in the extraction. Ultrasound in the
solvent producing cavitation accelerates the
dissolution and diffusion of the solute as well as the
heat transfer, which improves the extraction
efficiency. The other advantage of UAE includes low
solvent and energy consumption, and the reduction
of extraction temperature and time. UAE is
applicable for the extraction of thermolabile and
unstable compounds. UAE is commonly employed in
the extraction of many types of natural products.
Hydro distillation and
Steam Distillation
• Hydro distillation (HD) and
steam distillation (SD) are
commonly used methods for
the extraction of volatile oil.
Some natural compounds
encounter decomposition in
HD and SD.
•
Enzyme Assisted Extraction (EAE)

• The structure of the cell membrane and cell wall, micelles formed by
macromolecules such polysaccharides and protein, and the coagulation
and denaturation of proteins at high temperatures during extraction are
the main barriers to the extraction of natural products. The extraction
efficiency will be enhanced by EAE due to the hydrolytic action of the
enzymes on the components of the cell wall and membrane and the
macromolecules inside the cell which facilitate the release of the natural
product. Cellulose, α-amylase and pectinase are generally employed in
EAE.
Pulsed electric field
(PEF) extraction
• Pulsed electric field extraction
significantly increases the extraction yield
and decreased the extraction time because
it can increase mass transfer during
extraction by destroying membrane
structures. The effectiveness of PEF
treatment depends on several parameters
including field strength, specific energy
input, pulse number and treatment
temperature. PEF extraction is a non-
thermal method and minimizes the
degradation of the thermolabile
compounds.
Microwave assisted Extraction

• Microwaves generate heat by interacting with

polar compounds such as water and some organic
components in the plant matrix following the
ionic conduction and dipole rotation mechanisms.
The transfers of heat and mass are in the same
direction in MAE, which generates a synergistic
effect to accelerate extraction and improve
extraction yield. The application of MAE provides
many advantages, such as increasing the extract
yield, decreasing the thermal degradation and
selective heating of vegetal material. MAE is also
regraded as a green technology because it
reduces the usage of organic solvent. There are
two types of MAE methods: solvent-free
extraction (usually for volatile compounds) and
solvent extraction (usually for non-volatile
compounds)
Chromatography
1. Define chromatography
2. Explain classification of chromatography
technique
3. Explain type of phase in chromatography
4. Describe about paper chromatography
(definition, principle, method & their application)
in pharmaceutical industry.
 DEFINITION

CHROMATOGRAPHY

FThe separation of a mixture by distribution of its

components between a mobile and stationary phase
over time
• mobile phase = solvent
• stationary phase = column packing material
 HISTORY
• Chromatography
(from Greek :chromatos -- color , "graphein" -- to write)
• 1903 Tswett - plant pigments separated on chalk columns
• 1931 Lederer & Kuhn - LC of carotenoids
• 1938 TLC and ion exchange
• 1950 Reverse phase LC
• 1954 Martin & Synge (Nobel Prize)
• 1959 Gel permeation
• 1965 instrumental LC (Waters)
Purpose of
Chromatography
Analytical - determine chemical
composition of a sample

Preparative - purify and collect one

or more components of a sample
Uses for
Chromatography
Real-life examples of uses for chromatography:

Pharmaceutical Company – determine amount of each chemical found in

new product

Hospital – detect blood or alcohol levels in a patient’s blood stream

Law Enforcement – to compare a sample found at a crime scene to samples

from suspects

Environmental Agency – determine the level of pollutants in the water

supply

Manufacturing Plant – to purify a chemical needed to make a product

Classification There are two classification schemes:
• mobile phase
of Methods • attractive forces
 Gas (GC)

Water (LC)
Mobile Phase
Organic solvent (LC)

Supercritical fluid (SCFC)

Classification based on Mobile Phase

Gas Chromatography

Gas - solid Gas - liquid

Stationary Phase
Classification based on Mobile Phase

Liquid chromatography (LC)

Column Thin layer

High performance
(gravity flow) (adsorption)
(pressure flow)
 Adsorption

Classification Ion Exchange

based on
Attractive
Partition
Forces

Size Exclusion
Adsorption Chromatography

ØSeparation based on their

adsorption onto the surface of
solid (stationary phase).

Ø Normal phase-like separation

– Nonpolar mobile phase

Øfor polar non-ionic compounds

ØEx; Column chromatography

(CC) (K.Turus), TLC, HPLC
 Partition Chromatography
Øsolute are separated based on their partition
between a liquid mobile phase and a liquid
stationary phase coated on a solid support.

• Normal – analyte is nonpolar organic;

stationary phase MORE polar than the
mobile phase

• Reverse – analyte is polar organic; stationary

phase LESS polar than the mobile phase
• Ex : TLC, Paper Chromatography

Phase 2 Phase 2

Phase 1 Phase 1
Ion Exchange Chromatography
Ø Use ionic stationary phase
• ions separated on the basis of their tendency to
displace counter ions adsorbed on stationary phase
(Depends on charge, hydration, “solubility”
• Used for analysis of aminoacids and its base pair.

Ø Anionic stationary phases: used for cation separation

Ø Cationic stationary phases : for anion separation
Ø for ionic compounds
Ø - Ex : CC (K.turus), HPLC
Size Exclusion Chromatography
ØSeparation is a result of “trapping”
of molecules in the pores of the
packing material

• Very large molecules can’t get into

the pores – unretained

• Very small molecules get hung up in

to pores for a long time - most
retained – longest retention time

• stationary phase is a porous matrix

• Ex: CC, HPLC

Types of Chromatography
• Liquid Chromatography – separates liquid samples
with a liquid solvent (mobile phase) and a column
composed of solid beads (stationary phase)

• Gas Chromatography – separates vaporized samples

with a carrier gas (mobile phase) and a column
composed of a liquid or of solid beads (stationary
phase)

• Paper Chromatography – separates dried liquid

samples with a liquid solvent (mobile phase) and a
paper strip (stationary phase)

• Thin-Layer Chromatography – separates dried liquid

samples with a liquid solvent (mobile phase) and a glass
plate covered with a thin layer of alumina or silica gel
(stationary phase)
STATIONARY PHASE

Type of chromatography Material

Paper chromatography
(KK = kertas kromatografi)
Thin Layer Chromatography
(KLN = Kromatografi lapisan
nipis)
Gas chromatography
(GC)
High Performance Liquid
Chromatography
(KCPT = kromatografi cecair
prestasi tinggi)
Filter paper, cellulose
Silica gel, alumina,
polyamide
Squalene, apezion,
carbowax M
C-8, C-18, Licosorb,
Silicone
 MOBILE PHASE
Type of chromatography Solvent

Paper chromatography Air, alcohol

(KK = kertas kromatografi)
Thin Layer Hexane, ether petroleum,
Chromatography alcohol.
(KLN = Kromatografi
lapisan nipis)
Gas chromatography He, Ar, N2
(GC)
High Performance Liquid Cyclohexane, n-hexane,
Chromatography carbon tetrachloride, ethanol,
(KCPT = kromatografi methanol, air
cecair prestasi tinggi)
 PAPER
CHROMATOGRAPHY
 A chromatographic analytical separation
technique for complex mixtures involving
DEFINITION the progressive adsorption of the
dissolved component onto a special grade
of paper.
 PRINCIPLE

• The certain solvent are used to separate a

mixture ex: water, alcohol.

• With capillary action the solvent will move up

to filter paper.

• Movement of a solvent will bring together

component that are separated from the
mixture.

• Every component that are separated will move

to several velocity
The moving components depends on :
a. Solubility of solute in solvent
b. Intermolecule forces
c. Pore size of filter paper
d. Size of solute

At the end of process, components that are separated

will emerge to different distance on filter paper.

Rf values are used to identification of each the

component.
• The retention factor, or Rf, is defined as the distance
traveled by the compound divided by the distance
traveled by the solvent

For example, if a compound travels 2.1 cm and the

solvent front travels 2.8 cm, the Rf is 0.75:
 Materials List
• Beakers or jars
• Covers or lids
• Solvent (Distilled H2O,
Isopropanol)
• Graduated cylinder
• Filter paper
• Sampel (Different colors
of pens, plant extract)
• Pencil
• Ruler
• Scissors
• Tape
Preparing the solvent solution

• Prepare the solvent solution in various concentration:

- 0%, 5%, 10%, 20%, 50%, and 100%
 Preparing the Chromatography Strips

1. Cut filter paper

2. Draw a line 1 cm above

the bottom edge of
the strip with the
pencil

3. Label each strip with

its corresponding
solution

4. Place a spot from each

pen on your starting
line
Developing the Chromatograms

1. Place the strips in the beakers

2. Make sure the solution does not
come above your start line
3. Keep the beakers covered
4. Let strips develop until the
ascending solution front is
about 2 cm from the top of the
strip
5. Remove the strips and let them
dry
Developing the Chromatograms
Spot Detection
 1. Separation of ink dyes
- To compare ink dyes use in
any company.
2. Food coloring
- To differentiate coloring
USES OF PAPER agent used in food product
CHROMATOGRAPHY such as : M&M, Smarties dan
Reese candies.
3. Botanist/herbalist
- To isolate plant pigment from
root and leaves
Column
Chromatography
 • Wet Packing Technique
• » ideal & common technique
• The material is slurried with solvent and
generally added to the column in portions.
• S.P settles uniformly & no crack in the column of
adsorbent.
• » solid settle down while the solvent remain
upward.
COLUMN • » this solvent is removed then sea sand is
placed.
CHROMATOGRAPHY • The stationary phase settles uniformly in the
column and there is no entrapment of air
bubbles.
• There will not be any crack in the column of
adsorbent.
• The bands eluted from the column will be
uniform and ideal for separation.
 COLUMN
CHROMATOGRAPHY
Introduction of the Sample
• The sample which is usually a mixture of components
is dissolved in minimum quantity of the mobile phase.
• The entire sample is introduced into the column at
once and get adsorbed on the top portion of the
column.
• From this zone, individual sample can be separated by
a process of elution.
COLUMN
CHROMATOGRAPHY
COLUMN
CHROMATOGRAPHY
 least polar

heptane
cyclohexane, hexane
diethyl ether (ether)
benzene
increasing polarity, toluene
elution strength dichloromethane (DCM, MC)
ethyl acetate (EA)
alcohols (MeOH, EtOH)
chloroform
acetone
water
organic acid

most polar
 COLUMN
CHROMATOGRAPHY
• Development technique ( Elution)
• By elution technique, the individual components are
separated out from the column. The two techniques
are:
• (i) Isocratic elution technique : in this elution
technique , same solvent composition or solvent of
same polarity is used throughout the process of
separation. Iso means same/ similar.
• Example: chloroform only, petroleum: ether=1:1
 COLUMN
CHROMATOGRAPHY
(ii) Gradient elution techniques:

Ø Solvents of gradually ↑ polarity or ↑ elution strength

are used during the process of separation.
Ø Initially low polar solvent is used followed by gradually
increasing the polarity.
Ø E.g. initially benzene, then chloroform, then ethyl
acetate then chloroform
•
COLUMN CHROMATOGRAPHY
Eluting the sample: Components a, b, and c separate as column progresses.

• Fractions can be collected in test tubes, vials, beakers, or Erlenmeyer

flasks.
• Recovery is done by collecting different fractions of mobile phase of
equal volume like 10ml, 20ml etc or unequal volume.
They can also be collected timewise i.e. a fraction for every 10min or
20min etc.The recovered fractions are detected by using different
techniques.
 COLUMN
CHROMATOGRAPHY
• DETECTION OF COMPONENTS
• If the compounds separated in a column chromatography
procedure are colored, the progress of the separation can simply
be monitored visually.

• If the compounds to be isolated from column chromatography

are colorless. In this case, small fractions of the eluent are
collected sequentially in labelled tubes and the composition of
each fraction is analyzed by TLC.
 COLUMN
CHROMATOGRAPHY
Analyzing the fractions:
• Analyze the fractions by thin-layer
chromatography
 COLUMN CHROMATOGRAPHY
• FACTORS AFFECTING COLUMN EFFICIENCY
1. Dimension of the column: column efficiency has been improved
by increasing length/width ratio of the column.
2. Particle size of column packing: separation to be improved by
decreasing the particle size of the adsorbent.
3. Activity of the adsorbent
4. Temperature of the column: The speed of the elution increases
at higher temperatures.
5. Packing of the column
6. Quality of solvents: solvents having low viscosities is giving better
results.
7. Pressure
 COLUMN
CHROMATOGRAPHY
APPLICATIONS
►Separation of mixture of compounds
►Purification process (removal of impurities)
►Isolation of active constituents
►Estimation of drugs in formulation
►Isolation of active constituents
►Separation of diastereomers
 COLUMN
CHROMATOGRAPHY
• Advantages of C.C
• » Any type of mixture can be separated
• » Any quantity of mixture can be separated
• » Wider choice of Mobile Phase
• » Automation is possible
• Disadvantages of C.C
• » Time consuming
• » More amount of Mobile Phase are required
• » Automation makes the techniques more complicated & expensi
 Types of Liquid Chromatography

(TLC) Paper Gravity Chrom. Flash Chrom. HPLC 1952 UPLC 2004
Chrom. Tsvett, 1903 1978
Chromatography - Classification
• Basis of shape
• Column Chromatography – Open column, flash, vacuum
• Planar Chromatography – TLC, HPTLC, OPLC, Centrifugal TLC
• Mode of Separation
• Adsorption (NPC, LSC)– separates molecules based on polarity, least polar
eluting first
• Partition - (RPC, LLC) – Separates molecules based on combination of
solubility parameters, partition coefficients, and polarity, most polar eluting
first
• Ion exchange – Separates molecules on basis of molecular charge
• Size exclusion (GPC, GFC) – separation based on molecular size, largest
eluting first
• Affinity – based on affinity with ligand
• Basis of Mobile Phase
• Liquid Chromatography – LLC, LSC
• Gas chromatography – GLC, GSC
Various forms of Chromatography
• Column Chromatography
• Prep Column Chromatography
• Flash Chromatography (FC)

• Vacuum liquid Chromatography (VLC)

• Ion Exchange Chromatography

• Gel Chromatography
• Gel Filtration (GFC)

• Gel Permeation (GPC)

• Pressure Liquid Chromatography

• Low-Pressure LC
• Medium Pressure LC (MPLC)
• High Pressure LC (HPLC)
• Normal Phase and Reversed Phase Chromatography
Flash Chromatography
 An air pressure driven
It is also called as
hybrid of medium
“Medium Pressure
and short column
chromatography”.
chromatography.
INTRODUCTION
Invented by An alternative to slow
Clark & Still of and often inefficient
Columbia University gravity-fed
in 1978. chromatography.

77
Ø Differs from the conventional technique in 2 ways:

ü Slightly smaller silica gel particles (40 –

63 µm) are used.

ü Due to restricted flow of solvent caused by the

small gel particles, pressurized gas (10-15 psi)
used to drive the solvent through the column of
stationary phase

The net result is a rapid “over in a flash”and high

resolution chromatography.

78
 TRADITIONAL COLUMN CHROMATOGRAPHY

q In the traditional column

chromatography system, the user fills
the glass columns with silica gel.

q The sample is placed on the top of the

column. column runs by simply gravity
flow
q The separation is very slow and is
restricted to an isocratic solvent
mixture.

q At the end of the run, the silica gel must

be removed, cleaned, dried and re-
Column Chromatography packed.

79
EVOLUTION TO THE FLASH
CHROMATOGRAPHY
q In the modern Flash Chromatography
system the glass columns are replaced
with pre-packed plastic cartridges.

q Faster flow rates can be achieved by

using a pump or by using compressed
gas (e.g. air, nitrogen, or argon) to push
the solvent through the column.

q Systems may also be linked with

detectors and fraction collectors.
q The introduction of gradient pumps
means quicker separations, less
solvent usage and greater flexibility.

80
PRINCIPLE OF FLASH CHROMATOGRAPHY.
q The eluent is, under gas pressure (normally nitrogen or compressed air)
rapidly pushed through a short glass column with large inner diameter.

q The glass column is packed with an adsorbent of defined particle size. The
most used stationary phase is silica gel 40 – 63 μm.

STATIONARY PHASE

MOBILE PHASE

81
INSTRUMENTATION

82
Ø Pump Systems
Ø Pump Controller
Ø Vacuum Pump/peristaltic Pump
Ø Sample Injection Systems
Ø Columns, Filling Sets & Column Va lves
Ø Precolumns
Ø Fraction Collector
Ø Detectors and Chart Recorders
Ø Computerize LCD Display

83
PROCEDURE FOR MICROSCALE FLASHCOLUMN
CHROMATOGRAPHY

84
then tamp it down before Another way to fill the column is to
scooping more out pour the gel into the column using
a 10 mL beaker

85
PRE-ELUTION OFCOLUMN

86
 LOADING OF THE SAMPLE.
1.Wet loading method.
2.Dry loading method.

1.The sample to be purified is dissolved in

small amount of solvent. This solution is
loaded onto the column.

2.Dissolve the sample in small amount of

solvent & add silica gel. Swirl the mixture until
solvent evaporates & only dry powder
remains. This powder is loaded on the top of
the column.

87
ELUTION THROUGH THECOLUMN

88
 ANALYSIS OF THEFRACTION
v If the fractions are colored, combine like-colored fractions, although TLC
before combination is usually advisable.
v If the fractions are not colored, they are analysed by TLC .
v Once the composition of each fraction is known, the fractions containing the
desired compound(s) are combined.

89
90
91
Biotag Releveris
17
DETECTORS USED IN FLASH CHROMATOGRAPHY

UV-DETECTOR
94
 • Compounds are often difficult to detect due to a
lack of chromophores or, in the case of natural
products, the compound absorbance is unknown.

• Evaporative Light Scattering Detection (ELSD) has

ADVANCED DETECTION long been used for High Performance Liquid
TECHNIQUESFOR FLASH Chromatography, but has only recently been
CHROMATOGRAPHY employed for Flash chromatography.

• All-Wavelength Collection allows the collection of

compound with unknown
• absorbance or collection in the presence of
interfering solvent absorbance.

95
 96

CURRENT TRENDS IN
FLASH
CHROMATOGRAPHY…
FEATURES OF GREEN FLASHCHROMATOGRAPHY
Optimal parameters for flow rate, run time, fraction volume, etc. will be
calculated and set automatically upon selecting a column on “Green
Flash” software. The default parameters will be shown in System
Setting window.
Software provides the maximum sample load information for the
selected column.
State-Of-The-Art Software Based On True Theory of Chromatography.
Sample Eluting Position and Resolution Can Be Fully Controlled for
Systems.
Automatic Method Setup for Reverse Phase Chromatography.
Parallel Detection of UV Detector and RI Detector or ELSD.

97
98
 ADVANTAGES
q Large quantities of the sample can be separated (0.5-
2g)

q Fast ( 1o to 15 minutes)

q Cost efficient

q If high resolution is required, flash chromatography is

carried out before HPLC to avoid contamination of the
expensive columns.

99
 • Flash Chromatography has various applications in
following fields

v SYNTHETIC CHEMISTRY
ü It is used as a tool to monitor the reaction progress
and

APPLICATIONS ü to isolate and identify a mixture’s compounds.

ü It is used for separation of Closely related organic
compounds (isomer).
ü It is used to purify ,collect and identify
the various aromatic compounds in a
semi-synthetic extracts.

Ø Mestranol Purification During Chemical Synthesis.

100
ü Amino modified silica is used with normal phase solvents and is better
suited for nitrogen heterocyclic purification.
ü It has received increased attention as a lead investigation and
optimization tool in drug discovery.

Ø Bile Acid Purification During Lead Generation in Drug Discovery.

Ø In Anti-malarial Drug Purification in Drug Discovery.

10
1
v BIOLOGICAL STUDY

ü It is used for Purification of Protected Peptides.

Ø Normal phase Flash chromatography purified the protected Peptides
segments and reversed phase Flash chromatography purified final free
peptides.
Ø This two step purification eliminated the traditional costly & difficult
HPLC purification step

10
2
v PHYTOCHEMISTRY

ü Flash chromatography is a very valuable technique in the field of natural

compounds research because it provides a fast and economical way to
separate important phytochemical constituents of complex plant
extracts.

1.Separation and Isolation of α-Santalol and β-Santalol from Sandalwood

Extraction

2 . Isolation and Purification of Chromophoric and Nonchromophoric

Compounds in Giant Knotweed Rhizome.

3. Isolation and Purification of Flavonoids from Ginkgo Biloba Leaves

Extract.

10
3
4 . Isolation and Purification of Ginsenosides from Red
Panax Ginseng Extract.

5. Isolation and Purification of Catechins from Green Tea

Extract .

6 .In Purification of Galla Chinensis.

7. Purification of Ferulic Acid in Rhizoma Chuanxiong

Extract

10
4
The End.
Natural Products From Microorganisms
Part-1/2
Nur Athirah Yusof, PhD
Biotechnology Research Institute
 01 INTRODUCTION OF MICROBES
Bacteria, Archaea, Fungi, Virus

02
SPECIAL FEATURES OF MICROBIAL

WHAT ARE WE METABOLITES

GOING TO 03
BACTERIA AS SOURCE OF ANTIMICROBIAL
PROTEINS

COVER 04 MICROBES AS SOURCE OF ANTI-FUNGALS

05 ANTICANCER AGENTS
 1) Microbes
✓ Bacteria
✓ Archaea
✓ Fungi
✓ Viruses
2) Characteristics of microbes

OVERVIEW 3) The definition of microbial

metabolites

4) Special features of microbial

metabolites
MICROBES START
 WHAT ARE MICROBES?

o Microbes are organisms that can only be seen using a microscope.

o Microbes are widespread in nature and are beneficial to life, but some can
cause serious harm.

o They exist as unicellular, multicellular, or cell clusters.

o They can be divided into six major types: bacteria, archaea, fungi, protozoa,
algae, and viruses.
 BACTERIA
o unicellular organisms

o exist in four major shapes: bacillus (rod shape), coccus (spherical shape), spirilla (spiral
shape), and vibrio (curved shape)

o can be classified as either Gram-positive or Gram-negative

o divided based on their response to oxygen into the following groups: aerobic (living in the
presence of oxygen), anaerobic (living without oxygen), and facultative anaerobes (can
live in both environments)
E. coli image
o classified as heterotrophs (obtain their energy by consuming other organisms),
autotrophs (make their own food by using the energy of sunlight or chemical reactions),
saprophytes (use decaying life forms as a source of energy)
 gel-like matrix
composed of water,
enzymes, nutrients,
wastes, and gases
and contains cell
structures such as
ribosomes, a
chromosome, and
plasmids
hair-like structure for
attachment
contains all or most of
the genetic material

composed of a
phospholipid bilayer
acting as a
permeability barrier

made of
peptidoglycan to
maintain the shape

BACTERIAL
STRUCTURE polysaccharide layer
outside the cell
envelope
long, thin, whip-like
appendages for
movement
 BACTERIALSHAPES

BACILLUS (ROD SHAPE) COCCUS (SPHERICAL SHAPE) SPIRILLA (TWISTED SHAPE) VIBRIO (COMMA SHAPE)
Image: Bacillus anthracis Image: Staphylococcus aureus Image: Leptospira interrogans Image: Vibrio cholerae

Credit: CDC, 2009 Credit: Janice Haney Carr, Matthew J. Credit: CDC/NCID/HIP/Janice Credit: ktsdesign / Shutterstock
Arduino, DRPH, USCDCP Carr (PHIL #1220)
 ARCHAEA
o single-celled prokaryotic organisms that have distinct molecular characteristics
separating them from bacteria and eukaryotes

o discovered and described in extreme environments, such as hydrothermal vents

and terrestrial hot springs

o found in a diverse range of highly saline, acidic, and anaerobic environments

Thermal hot spring image

 EXTREMPHILICARCHAEA
ACIDOPHILES AND
THERMOPHILES
Image: Sulfolobus sp. A20
Growth conditions: pH 2-3,
75°C-80°C
Credit: Dai et al., 2016
ANAEROBIC AND
HYPERTHERMOPHILES
Image: Pyrolobus fumarii
Growth conditions: 95ºC to 113ºC
Credit: Blöchl et al. 1997
THERMOPHILES PSYCHROPHILIC METHANOGENS
Image: Thermococcus Image: Methanococcoides
gammatolerans burtonii
Growth conditions: 55°C and Growth conditions: 1-2°C
95°C, strongest known
Credit: Wikis
resistance to gamma rays
radiation
Credit: Angels Tapias, 2005
 FUNGI
o eukaryotic organisms

o may be either unicellular or multicellular

o Some are microscopic in size, while others form much larger structures
(mushrooms)

o do not contain chlorophyll

o absorb dissolved nutrients

Photomicrograph showing o can be found in the air, on plants and in water
the environmental form of Histoplasma
Credit: CDC, 2018 o cause a number of plant and animal diseases: in humans (ringworm, athlete's foot),
plant ( leaf, root, and stem rots)
 VIRUS
o Latin word of “poison”

o Obligate intracellular parasites (depend on the biochemical machinery of the

host cell for replication)

o Distinguished from bacteria by being “filterable agents”

o are not considered living (can't reproduce by themselves without a host & nor
do viruses have cells)
COVID-19 virus o Contain nucleic acid genomes, genetic variation and can evolve →
questionable!
HOW ARE VIRUSES DIFFERENT
FROM BACTERIA?
o bacteria are small and single-celled, but they are
living organisms that do not depend on a host
cell to reproduce.

o bacterial and viral infections are treated very

differently (antibiotics only work in bacteria)

o diameter of a typical virus is about 20- 300 nm,

average bacteria size 0.2 and 2.0 micrometer
 CHARACTERISTICS OF VIRUSES
Acellular infectious agents

Obligate intracellular parasites

Possess either DNA or RNA, never both

Replication is directed by viral nucleic acid

within a cell

Do not divide by binary fission or mitosis

Head-tail
Depend on host cell ribosomes, enzymes, and Credit Nossedotti (Anderson Brito),
nutrients for protein production Creative Commons Attribution

Lack genes and enzymes necessary for

energy production

Several viruses can cross the placenta &

cause developmental disturbances.
COMPONENTS OF MATURE VIRUSES (VIRIONS):
Either RNA or DNA, not both

Protein coat made up of

many protein subunits
(capsomeres). Capsomere
proteins may be identical or
different.

Consists of proteins, glycoproteins, and

host lipids. Derived from host membranes.
 WHAT ARE ◼ A variety of organisms, such as bacteria and fungi produce
secondary metabolites, also known as natural products.

MICROBIAL
◼ Microbial metabolites are chemical or carbon compounds
isolated from microorganisms.

METABOLITES
◼ The secondary metabolites may have antimicrobial, anti-tumour,
or anti-viral properties and anticancer.

◼ Antibiotics and other bioactive microbial metabolites are

produced by both prokaryotes and eukaryotes.
 Characteristics of the bioactive microbial metabolites:

SPECIAL ◼ origin

◼ interaction with the environment

FEATURES OF ◼ unique chemical structures

MICROBIAL In general, natural products including the microbial metabolites

may be practically utilized in three different ways:

METABOLITES ◼ Applying the natural/fermentation product directly in the

medicine, agriculture, or in any other fields.

◼ Using as starting material for subsequent chemical or

microbiological modification (derivatization).

◼ They can be used as lead compounds for chemical synthesis of

new analogs or as templates in the rational drug design (RDD)
studies.
THANK YOU!
ANY QUESTIONS?

nrathirah.yusof@ums.edu.my
Natural Products From Microorganisms
Part-2/2
Nur Athirah Yusof, PhD
Biotechnology Research Institute
 01 INTRODUCTION OF MICROBES
Bacteria, Archaea, Fungi, Virus

02
SPECIAL FEATURES OF MICROBIAL

WHAT ARE WE METABOLITES

GOING TO 03
BACTERIA AS SOURCE OF ANTIMICROBIAL
PROTEINS

COVER 04 MICROBES AS SOURCE OF ANTI-FUNGALS

05 ANTICANCER AGENTS
 INTRODUCTION
NATURAL PRODUCTS FROM
MICROBES
 NATURAL PRODUCT
APPLICATIONS
o Natural products originate as secondary metabolites from a myriad of sources,
including terrestrial plants, animals, marine organisms, microorganisms, terrestrial
vertebrates and invertebrates

o bio-chemicals ranging from alcohol to antibiotics and in processing of foods and

feeds

o natural sources of drugs for the treatment and prevention of diseases like cancer

o potential sources of natural antioxidants, colours, immuno-suppressants, enzyme

inhibitors, hypocholesterolemic agents, vitamins, enzymes, and antibiotics

o discovery of penicillin from Penicillium notatum (1928) by Alexander Fleming,

streptomycin (Streptomyces griseus), chloramphenicol (Streptomyces venezuelae),
etc.

o Lactococcus lactis- cell factories for the production of important nutraceuticals

o Lactobacillus acidophilus -triggered production of a beneficial immune molecule

o Probiotic bacteria – Lactobacillus, Bifidobacterium, Saccharomyces boulardii

o Prebiotics – induce growth of and the activity of useful microorganisms

Creative Commons Attribution 2.0
 DEVELOPMENT OF ANTIBIOTICS
o Antibacterial substances- hydrogen peroxide, fatty acids, organic acids,
ethanol, antibiotics, and bacteriocins.
o Antimicrobial peptides or proteins produced by bacteria are categorized as
bacteriocins.
o Bacteriocins are antibacterial peptides/proteins that either kill or inhibit the

ANTIBACTERIAL
growth of closely related bacterial strains.
o Generally only toxic to bacteria closely related to the producing strain

AGENT FOR FIGHTING o Example several Pediococcus strains can also be used effectively in food
systems to control Listeria monocytogenes

BACTERIAL o The killing ability of bacteriocins is to maintain population and reduce the
numbers of competitors to obtain more nutrients and living space in

INFECTIONS environments.
o Many bacteriocins are produced by food-grade lactic acid bacteria. Natural
preservatives to control the growth of spoilage and pathogenic bacteria in foods.
o Unlike most antibiotics, which are secondary metabolites, bacteriocins are
ribosomally synthesized and sensitive to proteases while generally harmless
to the human body and surrounding environment.
 MICROBIAL DERIVED ANTIBIOTICS

Yang, S. C., Lin, C. H., Sung, C. T., & Fang, J. Y. (2014). Antibacterial activities of bacteriocins: application in foods and
pharmaceuticals. Frontiers in microbiology, 5, 241. https://doi.org/10.3389/fmicb.2014.00241
DEVELOPMENT OF ANTIFUNGAL
◼ An antifungal agent is a drug that selectively eliminates fungal pathogens from a host with minimal toxicity to
the host.

◼ However, only a limited number of antifungal agents (polyenes and azoles, plus the recently introduced
caspofungin acetate) are currently available for the treatment of life-threatening fungal infections (Gupta et
al., 2014).
EXAMPLES FUNGAL INFECTIONS

RINGWORM ATHLETE’S FOOT FUNGAL NAIL INFECTION

Pictures credit: https://www.nhs.uk/

 NYSTATIN-ANTIFUNGAL
AGENT

• one of the first effective polyene

antifungal agent
• was obtained from Streptomyces
noursei
• Affective against Aspergillus
species
• topical antifungal agent in
treating oral, gastro-intestinal,
and genital candidosis

Picture of Aspergillus. Source of picture: https://www.cdc.gov/fungal/diseases/aspergillosis/definition.html

 DEVELOPMENT OF
ANTICANCER

• Microbial metabolites are among the

most important of the cancer
chemotherapeutic agents.
• There are many microbe-derived
anticancer agents that have been
evaluated through clinical trials.
• For instance, the polyketide
actinomycin was isolated from
Streptomyces parvulus in 1940 was
the first antibiotic shown to have
anticancer activity (Waksman and
Woodruff, 1940; Hollstein, 1974).

Breast cancer cell

MICROBIAL DERIVED ANTICANCERS

Gupta C, Prakash D, Gupta S. Natural useful

therapeutic products from microbes. J Microbiol
Exp. 2014;1(1):30‒37.
DOI: 10.15406/jmen.2014.01.00006
 NATURAL
COMPOUNDS AS
DRUGS ◼occur in low concentrations in nature

◼development burdensome

CHALLENGES? ◼economically impractical

 MICROBIAL HOSTS

For production of recombinant proteins and natural products

 More than 70% of FDA-approved
POINT-1 antibiotics are natural products, all of which
were derived from microbes.

Chemical diversity from microbial natural products

NATURAL POINT-2
leads to development of novel drugs ie antibiotic,
anticancer, and antifungal and along with other

PRODUCTS
pharmacological activities.

Microbial production of natural products involve

POINT-3 low production titers, difficulty in product isolation

FUTURE and structural identification and production of

inactive proteins.

PROSPECTS POINT-4 Engineering strategies to efficient production ie

genome editing, ribosome engineering, precursor
engineering, mutagenesis, and overexpression of
structural genes.

POINT-5 CRISPR/Cas as tools for genome editing for

additional improvements and to increase
production.
THANK YOU!
ANY QUESTIONS?

nrathirah.yusof@ums.edu.my