Kimia Medisinal

Drugs from
natural sources

Dwi Koko Pratoko, M.Sc., Apt

Pharmaceutical Chemistry
Department
Universitas Jember
 Mankind has explored natural resources
(abiotic and biotic) since early times and
has used them for survival and to develop
their civilization.

Plants are one of the biotic components in the biosphere which have been used
for food, clothing, ritual, medicine, dye, construction, cosmetics and others.

Extracts : medicine, dye, cosmetics

Natural products

Can be classified into three components, namely cellular structures,

primary metabolites, and secondary metabolites.

Natural products (sec. met) have a higher value in the market than
primary metabolites. Over 100,000 secondary metabolites
have been extracted from plants.

Secondary metabolites can be structurally divided

into alkaloids, phenylpropanoids, polyketides, and
terpenoids.
Basic biosynthetic pathways
Natural products: Isolation and
characterisation
The oldest attempt on natural product purification is distillation and
extraction process by decoction

In general, isolation starts from sample preparation

(pulverization), extractions-fractionation, and
separation.

Natural product separation was commonly conducted

using preparative
Molecular chromatographic
structure characterisation, methods
spectroscopic and spectrometric
(MS, NMR, UV, IR)
Natural products: Research and exploration
The study of plants is not new with evidence of studies since ancient times,
including the interaction between human-plants, plants-plants, and abiotic
environment-plants
Human-plants:

Food Phoenix spp (palm dates) was

domesticated back in the Late Middle
Paleolithic (300,000 to 30,000 years ago).

Medicine Achilles santolina has been used as

anti-dysentery since 60,000 years ago.
1981-2002: 868 new compound entities revealed from different sources

2008-2013: 100 natural products and natural product derived new entities
were in clinical trials which were investigated for potential for oncology
treatments (38%), anti-infective disease (26%), cardio-vascular and
metabolic disease (19%), inflammatory and related disease (11%), and
neurogical treatments (6%)
Plant-plant:
Allelophatic plantsRelease allelopathic chemicals into the immediate
environment which have an influence upon the growth and development of the
surrounding agricultural and biological ecosystem.

Weed management, Oryza sativa L (rice crop)

release momilactone A and B, effective against the
most harmful weeds in rice fields (banyrad grass:
Echinochloa crus-galli and Echinochloa colonum)
with concentrations of > 1 and > 10 M,
respectively.
Abiotic environment-plants:
These abiotic stresses included drought, temperature, UV-
radiation and CO2 level. Several plants produced
flavonoids (i.e anthocyanins) as a protective barrier
against drought and UV-radiation. A range of
cryoprotective compounds such as sugar alcohols were
synthesized as a reaction to cold during winter.
A study on plants grown in higher levels of CO2 revealed
changes in the plants chemical composition whereby the
synthesis of phenolics is increased and there is
condensation of tannins in leaves.
The impact of increasing the level of CO2 consistently
increased the level of phenolic compounds.
Drugs from natural resources
A. Scheme
1. Introduction
Potential Sources of new drug, novel lead compound, stereospecific
structures for the synthesis of existing drugs :
Plant
Animal
Marine
Common sources, plant and microorganism both land and marine
The selection may be based on ethnopharmacology or current
interest
Example:
South American Indians chewed coca leaves to alleviate
fatigue/tiredness cocaine.
2. Investigation

1st step : to decide its objectives

General purpose general compounds
Specific purpose bioactive compound
targets for specific diseases
Preparation sample
Screening assays
Fractionation & Isolation
Appropriate bioassay of compound
3. Bioactive isolated compounds
Molecular structure determination
Commercial interest synthesis and analogues synthesis
QSAR

Synthesis vs natural production

Economic efficiency cost of production comparison
Example:
Vincristine, vinblastine, etoposide, taxol

Catharanthus roseus
Vincristine
Firstly isolated in 1961
Chemotherapeutic agent
WHO essential medicine
Catharanthus roseus
Vinblastine
Firstly isolated in 1958
Chemotherapeutic
agent
WHO essential
medicine

Podophyllum peltatum
Etoposide
Chemotherapeutic
agents
4. Bioassay
Screening to detect the presence of active compound
in the initial extracts
Monitoring to follow the path of the active compounds
through the isolation process
useful to use screening and monitoring tests that can give
an estimate of the concentration of the active constituent
in the extract

The amount and degree activity of a constituent found

in naturally, depend on :
Environment
Time of collection
Preparation and storage
Screening test
Rapid, accurate and reproducible, high capacity, cheap
Tarry and poorly water soluble extracts
However, there is still possibile to miss active extracts
Broad or specific screening test (cytotoxic, anti-microbial,
etc)
Methods:
Whole organism screening test
i.e. Brine shrimp lethality test (BSLT), crown gall
tumour inhibition, bacteria, virus, fungi, etc
Cultured cell tests (mammalian cell)
Isolated enzyme tests
Isolated tissue tests
Cultured cell tests (mammalian cell)

Cell contain receptors that are specific to a

particular physiological process
These tests are used to study the binding of the
constituents of an extract to these receptors as
well as the inhibition of the action of that
receptor
Treatment of the cell culture with the extract is
normally followed by reaction with a suitable
reagent to produce a colour, fluorescence,
luminescence or radioactivity related to the
quantity of compound bound to the receptor or a
metabolite produced by the cell
 400 nm
Example, isolated enzyme tests trypsin
inhibition
Indicate debriding agents used to clean necrotic
wounds, ulcers and abscesses while inhibition
could show the presence of antithrombotic
agents, anti-inflammatory agents and male
antifertility agents
Other enzymes : alpha glucosidase, tyrosin
kinase etc
Isolated tissue test,
Ex : measuring contraction / inhibition contraction of a
suitable tissue (guinea pig ileum)
Monitoring tests
Concentration of active compounds increases during
fractionation
Activities might be fall/disappear during fractionation
Activity changes due to decomposition (nature of
process or inherent compound stability oxidation,
hydrolysis, polymerisation.
The removal of naturally occurring antioxidants, increases
the enzyme catalyse the decomposition, light exposure
Vigorous fractionation condition can be reduced by, low
temperature, solvent acidity-alkalinity as near neutral.
Solvent removed by either freeze drying or distillation
under low vacuum.
Activity changes due to synergy
Thalidomide is
more effetcive
Synergy mechanism????? Not understood in
yet. myloma
treatment in the
presence of
dexamethasone
 Activity change due to fractionation loss
Locked up in the procedures. i.e Chromatographic
technique: active compounds strongly or permanently
absorbed in a solid stationary phase. Distillation: co-
sublimation, co-distillation leaving inactive concentrate

5. Dereplication
Technique to eliminate extracts that contain active constituents
that have already been isolated and characterised. Wasting time
and resources
Compared to database
Case: NCI 40,000 natural product extracts were screened from
1987-1992. 15% active extracts anti-HIV active extracts
conitained similar classes of active compound
Example of dereplication procedure in anti-HIV drug discovery
from plants
Known active polysaccharide (anti-
HIV)

Polyphenols/tannins were retained

known anti-HIV active
6. Molecular structure analysis of isolated
compounds
Must be pure
Small isolates hard to get crystalline
Pure based on HPLC/GC/TLC
7. Active compound development
Naturally occurring active compounds into commercially
viable drugs

Cortisone extracted from the adrenal glands of cattle and

eventually
Synthesis 30 step
More efficient semiysinthetic methods based on progesterone
produced from diosgenin isolated from Mexican yams
B. Extraction procedures
Initial step
Separate desired chemicals from unwanted cellular debris and
chemicals i.e lipids, proteins, polysaccharides.
Need cleaning up
Design : objectives, source, scale, screening assay, chemical
compound in the extract (Polarity, acidity, alkalinity the
solvent)
 Acidity & Alkalinity

May affect the stability of the compound being extracted

Esters and amides hydrolysis in acidic/basic (esp if
extraction above RT)
1. Commonly used methods of extractions
Solvent-based methods
Infusion (ex : reflux distillation, soxhlet)
Steam distillation (to extract Essential oils)
Supercritical fluid extraction (ex : CO2)
Supercritical fluids are used for extraction because
the diffusion coefficients of solutes are higher than
would normally be expected for a liquid
their penetration and mass transfer of constituents
from natural materials is usually good
 Taxol were extracted using CO2 SFE from Catharanthus
roseus
2. Cleaning up procedures
Constituents fractionation difficulties, interfering
bioassay & isolation.
i.e. Tannisn, carotenes, chlorophyll bioassay problem.
Tannins cross link with protein, bioassay & isolation
problem.
Inorganic salts, i.e NaCl hard to be removed
Removal good and bad depends on the activity
3. Fractionation methods
the separation of an extract into groups of compounds
(fractions) having the same physicochemical characteristics,
such as solubility, size, electrical charge, acidic and basic
nature
Liquid-liquid partition partition (distribution) of a solute
between two immiscible liquids
Extraction of an extract is not limited to one extracting solvent
petroleum ether (low polarity), chloroform, ethyl ethanoate and
ethanol (high polarity), whereas a series with decreasing
polarity would be water (high polarity), water/ethanol (1:2),
ethanol and diethyl ether (low polarity)

Acidic and basic compounds

fractionations
 Mechanical methods
Automated liquid-liquid partition
Craig counter-current distribution (CCCD)

Salting out
adding very soluble inorganic salts, i.e NaCl, NH4Cl
into aqueous fraction.
This will reduce solubility of non-ionic compounds in
water and enhance their solubility in any less polar
immiscible slovent.
If very large quantities of the inorganic salt are used
and no other solvent is present, non-ionic compounds
may precipitate from solution
4. Chromatographic methods
separation techniques are the most widely used methods of
fractionation
Both thin-layer chromatography (TLC) and all forms of
column chromatography are used as fractionation
techniques
PTLC
5. Precipitation
Salting out widely used to isolate peptides and proteins
from solution
Solubility Reduction achieved by the addition of a second
miscible solvent in which the compound is less soluble
Insoluble salt formation way of regenerating and isolating
the required compound from the salt after separation of the
salt from the solution by either centrifuging or filtration
6. Distillation
used to separate volatile compounds
have a limited use and are normally only used to separate
essential oils from plant material
7. Dialysis
used to separate small water soluble compounds (<1000
Da) from larger molecules in aqueous solution
Small molecules will pass through the pores in a
semipermeable membrane when a concentration gradient
exists across that membrane
Dialysis is also used as a cleaning up procedure to remove
inorganic salts from extracts
8. Electrophoresis
used to separate components that carry an electrical
charge
Case story of
taxol
(Paclitaxel)

Taxus brevifolia (Pacific yew)

Taxol
1955 NCI screenings (synthesis
and natural products) Jonathan E
Well
1958 plant screenings (1000
species a year)
1962 Pacific yew collection
1964 extract, active
1968 1200 kg bark 28 kg crude
extract extract 10 g pure taxols
1975 anti-cancer in vitro (3,000 kg
bark)
1978 in vivo active (leukemic
mice)
1980 clinical (9,000 kg bark)
 1962 650 extracts investigated in Res. Triangle institute (Dr.
M.E. Wall under NCI contract
Extracts were positive against 9KB cell in vitro and L-1210
mouse in vivo
Taxus brevifolia 12 Kg was 95 percent methanol extracted
Biological assay against the Walker-256 solid tumour (5WM)

Partition (water and Chloroform methanol (4:1))

Organic layer 146 g solids showed good activity against 5WM
Solid applied into CCCD
Yield 0.004 percent
Low yield, solubility problem, limited bark supply
Three mature pacific yew tree to produce 1 g taxol
Asymmetric centres difficult to synthesis
Insoluble in water administration problem as most
chemitherapeutic use intravenous
Problem counteration
Polyethoxylated caster oil and absolute alcohol
solublise in 5% dextrose or saline
 1977 preclinical started
1979 mechanism of action (cell division)
1982 phase I trials
1985 phase II trials
1992 FDA approved 32 years after the discovery

Currently produced using semisynthetic pathway from baccatin III

and 10-deacetylbaccatin III (isolated from Leaves other Taxus).
No need for tress destruction.