NATURAL PRODUCTS

NATURAL PRODUCTS
Definition
Chemical substances produced by living organisms i.e.
produced during metabolisms process.

Sources
 Plants

 Microorganisms (bacteria and fungi)

 Animals

 Marine organism/microorganisms
NATURAL PRODUCTS: TRADITIONAL VS
CONVENTIONAL MEDICINE
CONVENTIONAL
 Single bioactive metabolite for
pharmaceutical formulation
 Tropical rainforest cover only
12% of earth’s land area, but
constitute 50-70% of world
species
 Less than 1% of tropical
rainforests species tested, but
at least 25% of modern drug
from rainforests
 Bioactive metabolites from
plants from tropical rainforest
formulate as drug or precursor
for drug
(Kong et al., 2003)
TRADITIONAL
 Standardized crude extract
 Phytochemical analysis
 Physical analysis
Safety assessment

Dosage performance

 80% world population depend on
alternative system of medicine
 Phytochemical studies of
medicinal plant resulted isolation
of important drug
 Cantharantus roseus originally
investigated for hypoglycemic
agents-yielded isolation
important anticancer drug (Vinca
alkaloid)
(Daniel, 2006; Newman et al., 2008)
 PRIMARY AND SECONDARY METABOLITES
Primary metabolites:
Essential for living organism

Similar metabolisms in all organisms with slight variations

• saccharides, amino acids, peptides etc

Secondary metabolites:
low molecular mass natural products and basically non essential for basic
metabolic processes of the plant

Secondary metabolites functions in plants as survival and adaptational

mechanism (Daniel, 2006).

 products derived from the above.

• having biological functions (known and unknown)
• usually small < 2000 mass unit.

(Daniel, 2006, Dewick, 2001).

SECONDARY METABOLITES FROM PLANTS
 Survival and adaptational mechanism
 Quinones-Intermediates in cellular respiration and photosynthesis

 Taxonomic evidence
 Occurrence restricted to certain families or genera

 Unique structural diversity

 Selective and specific biological activity

 Important lead compounds

 Vincristine, Vinblastine, Mitoxantrone

 Precursor drugs
 Vindesine (derivatives of vinblastine)

(Daniel, 2006; Hostettman, et al., 2000; Hong, et al., 2005)

THE FUNCTIONS OF SECONDARY
METABOLITES IN PLANTS
Secondary Metabolites Function Mechanism
Defensive Deterring herbivores due to their
Alkaloids
agents bitter taste
Attracting insect for pollination and
Volatile oils Pheromones
protect the plants from microbes.
When present in resin, perform
Diterpenes and
Antimicrobial wound healing and antimicrobial
triterpenes
function.
Limonoids and Insect Protect the plants especially wood
quassinoids repellents from insect attack.
Protect cellular membranes and
Phenolics Antioxidants tissues containing lipids from
oxidation.
Anthocyanins and Pollinator Pigments which make flowers visible
flavanoids guides to ‘colour blind’ insects
Pigments, Intermediates in cellular respiration
Quinones
Respiration and photosynthesis

(Daniel, 2006; Koyama, 2006)

CLASSIFICATION OF SECONDARY METABOLITES:

• Class of compounds –
• Fatty acids
• Terpenoids – mono-, sesqui-, di-, tri-, sester-, etc
• Alkaloids – morphinoids, aporphinoids, indole, etc.
• Flavonoids – catechol, flavanones, flavones, cyanidines.
• Quinonoids – benzo-, naphtho, anthra- etc.

• Class of Biosynthesis –
• Shikimic acid pathway
• polyketide pathway mix pathway
• mevalonate pathway
• alkaloid, heteroaromatics etc.
B The building
block for
U B secondary
metabolites are
I L derived from
primary
L O metabolism

D C Metabolites from
fundamental
I K process i.e.
photosynthesis,
glycolysis and
N S Krebs cycle
provides
G biosynthetic
intermediates
 Unsaturated
fatty acids

Saturated
fatty acids

Aromatic
THE ACETATE polyketides
PATHWAY: FATTY Phenolic
ACID AND Compounds
POLYKETIDES

prostaglandins
Leukotrienes
THE ACETATE PATHWAY
 Aromatic amino
acids
Benzoic acids
Lignans and
lignins

Flavanoids
THE SHIKIMATE PATHWAY: and stilbenes
AROMATIC AMINO ACIDS
Coumarins
AND PHENYLPROPANOIDS

Flavonolignans Terpenoid
quinones

stryrylspyrones
 Terpenes Iridoids

THE MEVALONATE
AND DEOXYXYLULOSE
PHOSPHATE
PATHWAYS:
TERPENOIDS AND
STEROIDS

Terpenoids Steroids
 NATURAL PRODUCT: DRUG DISCOVERY

Lead identification Lead Optimization Lead Development

1. Isolation and 1. Medicinal 1. Pharmacology
identification chemistry 2. Toxicology DRUG
2. Target based 2.Combinatorial 3. Pharmacokinetics CANDIDATES
bioassay chemistry Clinical Trials
4. ADME drug
3. In vivo and in delivery
vitro assays
 INTERACTION OF MULTIDISCIPLINARY FIELD,
INVESTIGATING MEDICINAL AGENTS FROM HIGHER PLANTS

Plant SAR
selection
PHYTOCHEMIST/
MEDICINAL
SYSTEMIC PHARMACOGNOSIST
CHEMIST
BOTANIST Extraction, isolation,
Synthesis
structural elucidation

In vitro screening plant extracts

Bio-assay guided fractionation
In vivo screening of pure compounds

Pharmacologist AND/OR microbiologist and/or BIOCHEMIST

Pharmacological and/or chemotherapeutical and/or biochemical
evaluation
Determination of mode of action of active products

Pieters, L., & Vlietinck, A. J. (2005), Verpoorte, R. (1998)

PLANT
COLLECTION
AND
PROCESSING
 CRITERIA FOR PLANT
SELECTION

 Ethnopharmacology
based on traditional uses in folk
medicine

 Random screening
on site screening or large screening
program (dereplication)

 Chemotaxanomy
targeted for specific secondary
metabolites in certain taxa
 PLANT COLLECTION AND SELECTION
Plant collection and documentation

Alkaloid Phytochemical screening Flavonoid

Sample processing and extraction

Biological Activity Testing

Antioxidant
Antimicrobial
(DPPH)
 PLANT COLLECTION AND DOCUMENTATION

 SK1417/07 – SK1439/07 (UiTM 1 – UiTM 23); main trail to Gua Luas, UiTM
research station, Sg. Keneyam, Taman Negara, Pahang; 165 m asl; N040 31’
04.2’’, E1020 28’ 28.5’’; 12/7/2007.

 SK1440/07 – SK1454/07 (UiTM 24 – UiTM 38); off trail of main trail to Gua
Luas, UiTM research station, Sg. Keneyam, Taman Negara, Pahang; 165 m
asl; N040 31’ 04.2’’, E1020 28’ 28.5’’; 13/7/2007.

 SK1455/07 – SK1464/07 (UiTM 39 – UiTM 48); riverside of Sg. Keneyam,

Taman Negara, Pahang; 165 m asl; N040 31’ 04.2’’, E1020 28’ 28.5’’;
14/7/2007.

 SK1465/07 – SK1466/07 (UiTM 49 – UiTM 50); main trail to Gua Luas, UiTM
research station, Sg. Keneyam, Taman Negara, Pahang; 165 m asl; N040 31’
04.2’’, E1020 28’ 28.5’’; 14/7/2007.
PHYTOCHEMICAL ON-SITE SCREENING
FLAVONOID ALKALOID
TEST TEST
15g dried sample moistened
Methanolic Extract with 10% NH3
+
+
15ml CHCl3 (Left for 10 min)
Mg ribbon + conc HCl
+
Equal amount 5% HCl + drops
Within 3 min Mayer’s reagent
Sample processing and extraction
Plant Material Dried ground plant material

Crude Extracts Extraction

 BIOLOGICAL ACTIVITY TESTING
(ANTIOXIDANT : DPPH METHOD)
A test samples in methanol will be added to 5 l of DPPH solution to give
final concentration of 250, 125, 62.5, 31.25, 15.625, 7.8125 and 3.906
μg/ml) in microtiter plate

Mixture is incubated for 30 min at room temperature

Absorbance at 517nm

The difference in absorbance between sample and control expressed as

percent inhibition
 EXPEDITION OUTCOMES
Eg: From two expeditions, 76 plant species
from 25 families were collected

Rubiaceae Rosaceae Lauraceae Rutaceae Commelinaceae

Annonaceae Moraceae Ebenaceae Simaroubaceae Burseraceae

Lecythidaceae Ulmaceae Verbanaceae Melastomaceae Convolvulaceae

Myristicaceae Menispermaceae Myrsinaceae Sterculiaceae Guttiferae

Tiliaceae Euphobiaceae Leeaceae Celastraceae Pandanaceae

20 species contain alkaloids
High content detected in the species in the table below
Psychotria sp Lasianthus constrictus Urophyllum hirsutum
(Rubiaceae) (Rubiaceae) (Rubiaceae)
Chassalia cartaceae Polyalthia cauliflora Salacia grandiflora
(Rubiaceae) (Annonaceae) (Selastraceae)

Lasianthus constrictus
42 species contain flavonoid
High content detected in the species in the table below
Labisia Knema
Urophyllum sp
pumilla glauca
Baccaurea Leea Disoxylum
racemosa indica cauliflorum
Phyllagathis Myristica
rotundifolia cinnamomea

Baccaurea racemosa Leea

indica
 Antimicrobial assay:
140 crude extracts of plant parts tested against 12
gram-positive and gram negative bacteria.

Bacillus Enterococcus Eschericia Klebsiella

subtillis feacalis coli pneumonia
Proteus Pseudomonas Salmonella Serratia
mirabilis aeroginosa typhnimurium marcescens
Staphylococc Sterptococcus
S. epidermis S. pyogenes
us aureus pneumonia

Psychotria sp and Salacia grandiflora

showed broad spectrum antibacterial
activity while several others were more
selective against these bacteria.
 The antioxidant property was measured based radical
scavenging activity against DPPH radicals.
Out of 50 species, 16 species showed >80% inhibition at 250
μg/ml concentration.

Species Part Family IC50

Horsfieldia tomentosa Warb. Stem Myristicaceae 5.73
Microcos laurifolia (Hook. ex Mast.) Burret Stem Tiliaceae 13.6
Leaves 28.2
Prunus lamponga (Miq.) Kalkman Rosaceae
Stem 15.6
Gironniera parvifolia Planch. Stem Ulmaceae 14.6
Antidesma velutinosum Blume Stem Euphorbiaceae 5.9
Horsfiedlia polyspherula (Hook.f.) J. Sinclair var. Stem 1.6
Myristicaceae
sumatrana (Miq.) W.J. de Wilde Leaves 30.2
Knema glauca (Blume) Warb. Stem Myristicaceae 27.4
Leaves 22.4
Psydrax sp. Rubiaceae
Stem 30.2
Leaves 3.9
Psychotria malayana Jack Rubiaceae
Stem 29.7

Note: Strong < 30μg/ml, Medium 30-100μg/ml, Weak > 100μg/ml

Horsfiedlia polyspherula
(Hook.f.) J. Sinclair var. Psychotria malayana Jack
sumatrana (Miq.) W.J. de
Wilde
ISOLATION AND
FRACTIONATION
 FRACTIONATION AND PURIFICATION
Cold solvent extraction
PLANT MATERIAL Soxhlet extraction
Microwave assisted extraction
Accelerated solvent extraction

CRUDE EXTRACTS
Vacuum liquid chromatography
Medium pressure liquid chromatography
Column chromatography
Preparative HPLC
FRACTIONS Solid phase extraction

Column chromatography
Medium Pressure liquid chromatography
Radial chromatography
PURE COMPOUNDS
Preparative HPLC
Preparative TLC
HPTLC
OVERVIEW OF NATURAL PRODUCT RESEARCH

Single drugs
Spectroscopic
data on-line: Spectroscopic
- LC/UV synthesis Data off-line:
- LC/MS - UV,
- LCNMR PRODUCT - MS,
structure - NMR,
elucidation - IR

PLANT
EXTRACTS FRACTIONS PURE CONST’ toxicology
MATERIALS
extraction separation separation

BIOASSAY BIOASSAY structure

modification PRODUCT
Crude drugs
BIOASSAY
GENERAL APPROACHES ON ISOLATION OF
CONSTITUENTS:

• Chemical based:
o Known function of certain group of compounds.

• Bioactivity Based (Bioactivity Guided Isolation):

 Selection of assay systems
 Animal?; cell?; enzyme?; chemical?

 Selection of research materials

 Random, Taxonomic consideration, Chemotaxonomic
consideration, ethnomedicinal or all.
LIMITING FACTORS:
• Chemical
• Separation, purification, identification.
• Human resources, expertise.
• Instrumentation.
• Cost and speed in acquiring reagents.

• Biological (assay)
• Relevance, consistency, reliability.
• Costs and speed in acquiring reagents.
• Human resources and expertise.
CHARACTERIZATION OF
METABOLITES
SPECTROSCOPIC METHODS FOR STRUCTURE ELUCIDATION
APPROACHES TO STRUCTURE ELUCIDATION
APPROACHES INFORMATION

Molecular mass Mass

Fragmentation pattern Skeleton
Skeleton? Fragment
MS

Functional
group Chromophore Functional group
IR UV-Vis

8.24
8.21

8.12

7.91
7.88

7.62
7.62
7.59
7.59

7.28

7.20
7.17
Equivalent hydrogen Types of hydrogen
Integration Number of protons
Hydrogen pair

1.09

1.00

1.07

1.10

1.10
1H NMR ppm (t1)
8.00 7.50

189.13

181.83

154.02
152.73
146.22
134.62
134.02
131.10
127.76
127.14
125.46
120.99
116.12
115.63

56.38

22.03
CDCl3

Types of carbons- qurternary,

DEPT methyl, methine, methylene C=O, C9
C-OCH3, C2

C-OH, C1
OCH3/C2a

CH3/C6a

Number of carbons in a molecule

C=0, C10

13C NMR
150 100 50 0
ppm (t1)

H5/C6a
1a
H7/C6a
H O OH

2-Dimensional NMR Assignment of chemical shift

2a
H 9 OCH3 50
8 1
7 12 13 2

6 11 14 3
6a
5 4

HMQC, HMBC, COSY, NOSY, Confirmation of proton and

H3C 10
H

100
H4/C13
H O H
OCH3/C3
H3/C14 CH3/C7,C8

ROESY carbon assignment

CH3/C5
H5/C12 CH3/C11
H5/C11 CH3/C6
150
H4/C1 H3/C2 OCH3/C1

H5/C10 H4/C10
H8,C9
ppm (t1)

8.0 7.0 6.0 5.0 4.0 3.0

ppm (t2)
 1-hydroxy-2-methoxy-6-methyl-
9,10-anthraquinone
O OH

8 2a
1 OCH 3
7 9
12 13 2

11 14 3
6a 6 10
H3C
5 4

M/z 268 C16H12O4

UV EtOH: 421 nm, UV EtOH/OH: 505 nm Ismail, N. H., Osman, C. P., Ahmad, R.,
IR (KBr) :3467, 1653, 1637 Awang, K., & Ng, S. W. (2009). 1-Hydroxy-
2-methoxy-6-methyl-9,10-anthraquinone
from Rennellia elliptica Korth. Acta
Osman, C. P., Ismail, N. H., Ahmad, R., Ahmat, Crystallographica, E65, o1435.
N., Awang, K., & Jaafar, F. M. (2010).
Anthraquinones with Antiplasmodial Activity
from the Roots of Rennellia elliptica Korth.
(Rubiaceae). Molecules, 15(10), 7218-7226.
 13.15

O OH

4.04

2.56
8 2a
1 OCH 3
7 9
12 13 2

11 14 3
6a 6 10
H3C
5 4

C2a
OCH3

C6a
CH3
DCM residue Impurities

C1
OH
1.01

3.13

3.10
10.0 5.0
ppm (t1)
 8.24
8.21

8.12

7.91
7.88

7.62
7.62
7.59
7.59

7.28

7.20
7.17
O OH

8 2a
1 OCH 3
7 9
12 13 2

11 14 3
6a 6 10
H3C
5 4

O H4, J=8.4 Hz

H8,J=8.1 Hz
H5 H3,J=8.4 Hz
H7,J=8.1 Hz
1.09

1.00

1.07

1.10

1.10
8.00 7.50
ppm (t1)
COSY

7.00

7.50

8.00
8.23 13.20
H O OH

7.61 H OCH 3 4.04

2.56 H3C H 8.50

7.19
H O H
8.12 7.89 ppm (t1)

8.50 8.00 7.50 7.00

ppm (t2)
NOESY

2.50

8.23 13.20
H O OH
3.00
7.61 H OCH3 4.04

2.56 H3C H
7.19 3.50
H O H
8.12 7.89

4.00

ppm (t1)

8.00 7.50 7.00

ppm (t2)
 CARBON-13 NMR
189.13

181.83

154.02
152.73
146.22
134.62
134.02
131.10
127.76
127.14
125.46
120.99
116.12
115.63

56.38

22.03
CDCl3

O OH

8 2a
1 OCH 3
7 9
12 13 2

11 14 3
6a 6 10
H3C
5 4

OCH3/C2a

C-OCH3, C2 CH3/C6a

C=O, C9 C-OH, C1
C=0, C10

150 100 50 0
ppm (t1)
HMBC

H5/C6a
1a
H7/C6a
H O OH
2a
H 9 OCH3 50
8 1
7 12 13 2

6 11 14 3
6a
5 4
H3C 10
H

100
H4/C13
H O H
OCH3/C3
H3/C14 CH3/C7,C8
CH3/C5
H5/C12 CH3/C11
H5/C11 CH3/C6
150
H4/C1 H3/C2 OCH3/C1

H5/C10 H4/C10
H8,C9
ppm (t1)

8.0 7.0 6.0 5.0 4.0 3.0

ppm (t2)
MEDICINAL PLANTS
WHAT IS “MEDICINAL PLANT”?
 A plant that contains a substance or substances
which modulate beneficially the physiology of sick
mammals, and that it has been used by man for that
purpose.

What is a “weed”?
 A plant whose virtues have not yet been discovered

 Ralph Waldo Emerson

POTENTIAL OF TROPICAL RAIN FORESTS AS
SOURCE OF NEW THERAPEUTIC AGENTS
 Indonesia is the second biodiverse nation in the world
 Estimated no. of plant species in selected countries
 Market value of drugs/medicinal produced from plants is US
43 billion in 1989.

Country No. of Species

Malaysia 12 000
Indonesia 20 000
Philippines 10 000
USA 9 000
Brazil 55 000
Britain 1 550
Germany 2 600
PHARMACEUTICALS OF PLANT/
MICROORGANISM ORIGIN
Pharmaceuticals Sources
Codein Papaver somniferum
Reserpine Rauwolfia serpentine
Digoxin Digitalis lanata
Digitoxin Digitalis purpurea
Scopolamine Datura metel
Vincristine/vinblastine Catharanthus roseus
Cephalosporins Cephalosphorium sp.
Ephedrine Ephedra sinica
Quinidine Cinchona ledgerina
 Morinda elliptica
 Common names:
“Mengkudu kecil”

 Medicinal uses:
1.Loss of appetite, headache, cholera,
diarrhea and fever
2.Enlarged spleen and wounds
3.Hemorrhoid and after childbirth
O OH
15 O OCH3
8 1
12 9 CHO
7
13
2
CHO

6 3
11 10 14

5 4
OH
O
O

2-formyl-2- Damnacanthal
hydroxyAQ (new) *strong anti-HIV
*anticancer

Ismail, N. H., Ali, A. M., Aimi, N., Kitajima, M., Takayama, H., & Lajis, N.
H. (1997). Anthraquinones from Morinda elliptica. Phytochemistry,
45(8), 1723-1725.
 O

Cassia alata
 Common names:
Gelenggang besar
OH O OH

 Medicinal uses:
Chrysophanol
Laxative, ringworm, constipation,
intestinal worm
O
OH O OH

OH

OH
OH O OH
O

*Aloe-emodin
Emodin

*Anticancer,
antimicrobial
 Andrographis paniculata
 Common names:
sambiloto, hempedu bumi

 Medicinal uses:
liver disease, high blood pressure
O O

O O
HO

HO HO
H
OH OH

Andrographolide 1,4-deoxyandrographolide

Reddy, M. K., Reddy, M. V. B., Gunasekar, D., Murthy, M. M., Caux, C., & Bodo, B. (2003). A
flavone and an unusual 23-carbon terpenoid from Andrographis paniculata.
Phytochemistry, 62(8), 1271-1275.
 Centella asiatica
 Common names:
pegagan

 Medicinal uses:
liver disease, high blood pressure

Asiaticoside

Kim, W.-J., Kim, J., Veriansyah, B., Kim, J.-D., Lee, Y.-W., Oh, S.-G., et al.
(2009). Extraction of bioactive components from Centella asiatica using
subcritical water. The Journal of Supercritical Fluids, 48(3), 211-216.
Eurycoma longifolia Jack
 Common names:
pasak bumi, tongkat ali

 Medicinal uses:
aphrodisiac

1-eurycomanone
2-14,15β-Dihydroxyklaineanone
3-13,21-Dihydroeurycomanone
Chan, K.-L., Choo, C.-Y., Abdullah, N. R., & Ismail, Z. (2004). 4-13,21-Epoxyeurycomanone
Antiplasmodial studies of Eurycoma longifolia Jack using the 5-Eurycomanol
lactate dehydrogenase assay of Plasmodium falciparum. 6-Eurycomalactone
Journal of Ethnopharmacology, 92(2-3), 223-227. 7-9-Methoxycanthin-6-one
Study Field
at Purwodadi Forest
East Java