Title: NATURAL PRODUCTS: STRATEGIES AND IMPORTANCE IN DRUG DISCOVERY, AND THE MALAYSIAN ASPIRATIONS
1NATURAL PRODUCTS STRATEGIES AND IMPORTANCE IN
DRUG DISCOVERY, AND THE MALAYSIAN ASPIRATIONS
Prof. Dr. Khalijah Awang
- Chemistry Department
- Faculty of Science
- University of Malaya
- Malaysia
2WHAT ARE NATURAL PRODUCTS?
- Is everything found naturally (from nature ) a
natural product??? - Natural products are secondary metabolites
produced by an organism, small molecules (mol wt
lt 1500 amu approx.) But are not strictly
necessary for the survival of the organism.
3EXAMPLES OF NATURAL PRODUCTS
4NATURAL PRODUCTS AND DRUG DISCOVERY
- Plants as a form of medication
- 1803-Derosne extracted a mixture of narcotine and
morphine - 1806-Serturner recognised the alkaline nature of
the principle content of opium - However the structures of these complex molecules
were made known only in the 2nd half of the 20th
century - NMR X-ray- the tedious task has become much
easier.
5NATURAL PRODUCTS AND DRUG DISCOVERY contd
- Many drugs were discovered from natural products
and their complex structures have intrigued many
chemists and due to this, synthesis of them were
of great challenge. - 1952-morphine was synthesised.
- 1950s reserpine was synthesized.
6NATURAL PRODUCTS AND DRUG DISCOVERY contd
- Molecules structures morphine, reserpine,
vinblastine-vincristine, taxol. - Drug synthesis expensive so extraction and
hemisynthesis.
7NATURAL PRODUCTS AND DRUG DISCOVERY -contd
- Drug development cost has increased tremendously
800 million USD - 1990s combinatorial technology
- Synthesis high throughput screening to cut
cost - For about one decade natural products research
was dampened - But now industrialists realised the importance of
natural products diversity and biocompatibility
8NATURAL PRODUCTS AND DRUG DISCOVERY contd
- TO FIND NEW LEADS
- Natural products high throughput screening
- Natural product research is revived and
revitalized - Preliminary biological screening of samples from
living organisms plants and others - Bioassay guided studies of the bioactive extracts
- Structural elucidation
- Biological testing of the isolated compounds
- Sar studies
9PRELIMINARY BIOLOGICAL SCREENING OF SAMPLES
- Plant collection
- Identify the botanical name
- Prepare a herbarium sample
- Dry and grind sample
- Extract the samples with various solvents
- Send extracts for biological screening
10HERBARIUM SAMPLE
- Herbarium KL 4937
- Name Fissistigma manubriatum (HK. F. et Th.)
Merr. - Family Annonaceae
- Locality and habitat 57 km from Mersing towards
Kluang, Johore. - Field description
- A woody climber, main stem 5-7 cm diam, bark
brown to darkish brown young parts rusty
tomentous leaves thinly coriaceous, from oblong
to oblanceolate - Duplicate Kew, Leiden, Paris, K.L.
- Date 20 Apr. 2000
- Ident L. E. Teo Din
11BIOASSAY GUIDED STUDIES OF THE BIOACTIVE EXTRACTS
HUNTING FOR THE ACTIVE MOLECULE/S
- Fractionation of the active extracts
- Send fractions for biological screening
- Isolate compounds from active fractions
- Structural elucidation of isolated compounds
- Send pure compounds for biological screening
- Hopefully active molecule/s is/are found
12BIOASSAY GUIDED STUDIES OF THE BIOACTIVE EXTRACTS
contd
- Fractionation and isolation
- The right chromatography techniques CC, TLC,
VC, MPLC, HPLC solvent systems and stationary
phase critical in hunting down the bioactive
compound/s.
13MOST COMMON CHROMATOGRAPHIC TECHNIQUES
- CHROMATOGRAPHIC TECHNIQUES
- Column chromatography
- Thin layer chromatography
- MPLC
- HPLC
- STATIONARY PHASE
- Silica-nonpolar
- Bonded silica-C18, C-8, CN etc
- Polyacrylamide
- Polysaccharides sephadex
14STRUCTURAL ELUCIDATION
- Spectroscopic methods
- Infrared (IR)
- - indicates presence of functional groups
- CO 1670 1750 cm-1
- amide, ketone, ester
- OH, NH/NH2 3100 cm-1 to 3500 cm-1
- Limitation non polar and semi polar
- compounds only.
15STRUCTURAL ELUCIDATION contd
- Mass Spectrometry
- Enables the determination of molecular weight.
- Aids structural elucidation fragmentation
peaks loss of CO (M- 28), loss of H2O (M- 18).
- Enables identification of mixtures MS-MS.
- Various ionisation techniques to accommodate
different compounds polar, ionic, nonpolar,
macromolecules. - Various Analysers usage (MS-MS, HRMS), cost.
16COMMON MODES OF MS IONIZATION
- Electron Impact (EI)
- Chemical Ionization (CI)
- Fast Atom Bombardment (FAB)
- Thermospray Ionization
- Electrospray Ionization (ES)
- Atmospheric Pressure Chemical Ionization (APCI)
17COMMON MASS ANALYZERS
- Magnetic Deflection Mass Analyzer
- Double Focusing Sector Spectrometer
- Quadrapole Mass Analyzer
- Triple Quadrapole Analyzer
- Ion Trap
- Time of Flight Spectrometer
18MASS FRAGMENTATIONS OF ERYTHROCARPINE A
19NUCLEAR MAGNETIC RESONANCE
- Nuclear Magnetic resonance
- Permits the establishment of the structural
skeleton of the compound investigated. - 1HNMR showed resonances of protons while 13C NMR
showed the C resonances. - Able to establish the connectivity between
carbons and protons. - One dimensional and two dimensional techniques
available - COSY, HMQC, HMBC, NOESY etc.
- For 1HNMR 1-5 mg (pure) sufficient
- For 13C NMR 20 mg sufficient.
20lysicamine
norboldine
pallidinine
tetrahydropronuciferine
reticuline
O-methyldauricine
phoebescortechiniine
212-OMe
1-OMe
5
11
10
8
4
9
lysicamine
2210-OMe
1-OMe
8
3
11
norboldine
237-OMe
2-NMe
12-OMe
7-OMe
6-OMe
2-NMe
6-OMe
8 8
5 5
11 13
13 14
10 14
10
11
O-methyldauricine
246-OMe
3-OMe
-NMe
4
1
5
16
10ax
9
15
8
10ef
pallidinine
254-OMe
-NMe
2-OMe
1-OMe
2
5
2
3
1-NH
phoebescortechiniine
26reticuline
27-NMe
2-OMe
1-OMe
3
7
6a
12
tetrahydropronuciferine
28INACTIVE COMPOUNDS IMPORTANT?
- Today inactive but 10 years later ???
- Taxol (anticancer drug)
- Structure identified in 1971, mechanism of action
1979 - Rhazinilam (anti tubulin agent)
- Structure identified in 1972, anti-tubuline
(anticancer) activity discovered in 1990. - Biogenesis relationship between the compounds.
- Chemical relationship between the active compound
and major compound hemisynthesis - 10 deacetylbaccatine III ? taxol and taxotere.
29HEMI-SYNTHESIS OF TAXOL
30NEW NATURAL PRODUCTS DRUGS
31 NATURAL PRODUCTS ON CLINICAL TRIAL
32THE MALAYAN BORNEO FLORA
- One of the oldest and richest in the world
- More than 200 million years of age
- About 15,000-18,000 flowering species (Malayan
Borneo)
33FIELD TEAM (2000)
34HERBARIUM
35ANNONACEAE
Monocarpia
Fissistigma
Artabotrys
Cyathostemma
36LAURACEAE
Phoebe grandis
Phoebe lanceolata
Kryptocarya kurzii
Dehaasia sp.
37MELIACEAE
Aglaia malaccensis
Chisocheton tomentosus
Aglaia argentia
38APOCYNACEAE
Alstonia angustifolia
Kopsia pauciflora
Kopsia teoi
39HERBARIUM SAMPLE
- Herbarium KL 4937
- Name Fissistigma manubriatum (HK. F. et Th.)
Merr. - Family Annonaceae
- Locality and habitat 57 km from Mersing towards
Kluang, Johore. - Field description
- A woody climber, main stem 5-7 cm diam, bark
brown to darkish brown young parts rusty
tomentous leaves thinly coriaceous, from oblong
to oblanceolate - Duplicate Kew, Leiden, Paris, K.L.
- Date 20 Apr. 2000
- Ident L. E. Teo Din
40BIOACTIVE COMPOUNDS FROM THE MALAYAN FLORA
Argentinic acid A methyl ester
Forbesine
41rhazinilam
2R kurziflavolactone A 2S kurziflavolactone B
kurzilactone
42Pedicin
(-)- sebiferine
43(-)-sebiferine
Erythrocarpine D
(-)-0,0-dimethylgrisabine
44PRESENT WORK
- Huncaniterine A
- A new bisindole alkaloid from Hunteria
zeylanica Kg. Padang, Kuala Lipis, Pahang - Exhibited vasorelaxation effects on isolated rat
aorta - Showed responses against norepinephrine (3x10-7
M) - Bisbenzyl and secobisbenzyl isoquinoline
alkaloids - Phaeanthus crassipetalus Dungun, Terengganu
- Used traditionally in Borneo, especially Sabah,
for the treatment of wounds and high blood
pressure - 3 bisbenzyls and 3 secobisbenzyls that possess
antihypertensive activity
45PRESENT WORK-Contd
- Labdane diterpenes
- Andrographis paniculata
- 6 knowns labdane diterpenes were isolated and
have shown coronary vasodilation activity. - Limonoids
- Chisocheton erythrocarpus Hutan Simpan Terenas,
Kedah - Dichloromethane extract showed cytotoxic
activity against P388 murine leukaemia cells
(IC50 18mg/ml) - Six new and cytotoxic limonoids were isolated
and identified
46Hunteria zeylanica
Huncaniterine A (Hunteria zeylanica) Heterocycles
Vol. 74 (2007)
47ANTI-HYPERTENSIVE ALKALOIDS FROM PHAENTUS
CRASSIPETALUS
Phaeanthus crassipetalus Becc.
- Phaeanthus crassipetalus Dungun, Terengganu
- Only 2 species in Malayan Forest P.
crassipetalus P. ophthalamicus - Used traditionally in Borneo, especially Sabah,
for the treatment of wounds and high blood
pressure - 3 bisbenzyls and 3 secobisbenzyls that possess
antihypertensive activity
483 BISBENZYLS
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503 SECOBISBENZYLS
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52BIOASSAY GUIDED STUDIES ON ANDROGRAPHIS PANICULATA
- Family Acantaceae
- Local name
- Hempedu bumi
- King of bitter
- Hypertension, diabetes, malaria and fever
Andrographis paniculata
53BIOASSAY contd
The effects of hexane (HE), dichloromethane (DE)
and water (WE) extracts of Andrographis
paniculata, and sodium nitroprusside (positive
control) on coronary perfusion pressure in
isolated rat hearts. denotes p lt 0.05, paired
t-test extract versus its vehicle control). The
methanol extract did not show significant effects
on coronary perfusion pressure, data not presented
Group Basal coronary perfusion pressure (mmHg) Change in coronary perfusion pressure (mmHg) (mean S.E.M) Change in coronary perfusion pressure (mmHg) (mean S.E.M) Change in coronary perfusion pressure (mmHg) (mean S.E.M) Change in coronary perfusion pressure (mmHg) (mean S.E.M) Change in coronary perfusion pressure (mmHg) (mean S.E.M) Change in coronary perfusion pressure (mmHg) (mean S.E.M)
Group Basal coronary perfusion pressure (mmHg) Extract (mg) Extract (mg) Extract (mg) Extract (mg) Extract (mg) Sodium nitroprusside 1000nmol
Group Basal coronary perfusion pressure (mmHg) 0.001 0.01 0.1 1 3 Sodium nitroprusside 1000nmol
HE (n4) Vehicle 94.0 9.9 2.5 3.2 3.8 3.8 -5.0 2.9 -2.5 1.4 -17.5 5.2 3.8 5.9 -47.5 14.8 12.5 6.3 - - -43.8 14.1
DE (n5) Vehicle 96.4 12.7 -2.4 2.4 -2.6 2.6 -8.6 2.7 10.0 6.3 -14.0 4.8 -4.6 3.0 -29.4 8.5 -4.0 12.6 -24.5 3.0 12.5 7.9
WE (n4) Vehicle 85.0 20.2 0 0 0 0 -12.0 6.0 0 -27.3 11.8 0 -36.3 18.0 0
54BIOASSAY contd
- The results showed that hexane, CH2Cl2 and water
extract of the AP extracts tested were able to
modulate rat cardiovascular function directly. - Except for the methanol extract, all other
extracts caused coronary vasodilation, as shown
by reductions in coronary perfusion pressure. - The hexane extract appeared to be the most active
in this effect. In addition, the water extract
was able to increase cardiac contractility, as
observed from the increase in developed tension.
55BIOASSAY contd
andrographolide
14-deoxy-11,12-dihydroandrographolide
neoandrographolide
14-deoxy-12-hydroandrographolide
14-deoxydihydroandrographolide
56BIOASSAY contd
The effects of diterpenoids isolated from the
dichloromethane extract of Andrographis
paniculata, sodium nitoprusside (SNP) and 40
EtOH on coronary perfusion pressure of isolated
rat hearts. The results are expressed as mean
s.e.m., plt0.05, students paired T-test,
compounds versus vehicle (40 ethanol). Basal
coronary perfusion pressure 72.15.6 mmHg.
57BIOASSAY contd
- All of these diterpenoids reduced coronary
perfusion pressure (CPP) significantly but
compound 4 reduced CPP most (-19.5 7.0 mmHg).
- This value was comparable to sodium nitroprusside
(positive control), which reduced CPP by 22.0
5.4mmHg. The combination of all diterpenoids of
the same amount reduced CPP by 10.8 3.7 mmHg. - None of the five diterpenoids showed any
significant effect on heart rate even though the
positive control, isoprenaline, was effective.
58BIOASSAY GUIDED STUDY OF CHISOCHETON
ERYTHROCARPUS AGAINST P388 MURINE LEUKAEMIA CELLS
- Collected from Hutan Simpan Terenas, Kedah.
- 26/51 Chisocheton species in Peninsular Malaysia.
- The very first Malaysian species to be studied
chemically. - Dichloromethane extract IC50 18mg/ml
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62CYTOTOXIC ACTIVITY AGAINST P388 MURINE LEUKAEMIA
CELLS
Compounds IC50 (mg/ml)
Erythrocarpine A (1) 2.0
Erythrocarpine B (2) 6.0
Erythrocarpine C (3) 6.0
Erythrocarpine D (4) 9.9
Erythrocarpine E (5) 10.0
Erythrocarpine F (6) 16.0
MMC (mitomycin C) 0.15
63MALAYSIAN ASPIRATIONS
- Malaysia rich in both natural resources and
cultural heritage - Natural Resources megabiodiversity with our
Malayan and forests - Cultural heritage traditional medicines
- Malaysia is represented by three major ethnic
groups Malay, Chinese and Indian - Three different type of traditional medicine
practiced by all communities - Jamu (Malay)
- Sensei (Chinese)
- Ayurvedic (India)
64MALAYSIAN ASPIRATIONS Contd
- Malaysian aspiration in the natural products
research - To execute a thorough biological and chemical
studies on the Malayan and Borneo rain forests
(less than 5 of plant species have been
studied) - Chemotaxonomy
- Bioassay guided studies Lead discoveries
- Help to develop drug discovery program (with
foreign collaboration/pharmaceutical industries)-
very high cost - To study thoroughly the known medicinal
plants/herbs for the development of
nutraceuticals and public knowledge. - To upgrade the present traditional medicines/
herbal food supplements in the market
bioactivities, bio-assay guided,
standardization, toxicology and clinical studies.
- Help modernize the herbal industry to be
accepted globally - The population will benefit quality herbal and
pharmaceutical - products at affordable price.
65PROBLEMS OF NATURAL PRODUCT RESEARCH
- Lack of co-ordination and monitoring
- Projects should be well managed and monitored
- Better / stronger cooperation among researchers
and institutions - Documentation (Burkill)
- Industries (linkage)
- Lack of funding
- Human resource (expertise) / training
- Equipments / services
- Lack of focus
- Researchers-too many administrative
responsibilities - Training for staffs and students
- Regulations
- National Bereau of Pharmacy
66CHALLENGES
- Capitalise on our assets
- Overcome our problems professionally
- Think global not local
- Governmentresearchers industries better
communication and trust
67INSYAALLAH
- Malaysia will be a global force in natural
products research and pharmaceutical
/nutraceutical industries.
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74TERIMA KASIH THANK YOU