secondary metabolites from plants plant secondary metabolites

1

Secondary Metabolites from Plants

David S.-Y. Wang

Assistant Professor

Department of Forestry, NCHU

Plant Secondary Metabolites

Secondary metabolites are those metabolites which are often produced in a phase of subsequent to growth, have no function in growth (although they may have survival function), are produced by certain restricted taxonomic groups of microorganisms, have unusual chemicals structures, and are often formed as mixtures of closely related members of a chemical family.

The simplest definition of secondary products is that they are not generally included in standard metabolic charts.

2


A metabolic intermediate or product, found as a differentiation product in restricted taxonomic groups, not essential to growth and the life of the producing organism, and biosynthesis from one or more general metabolites by a wider variety of pathways than is available in general metabolism.

Secondary metabolites are not essential for growth and tend to be strain specific. They have a wide range of chemical structures and biological activities. They are derived by unique biosynthetic pathways from primary metabolites and intermediates.


Biochemical pathways that are not necessary for growth

or reproduction of an organism, but which can be

demonstrated genetically, physiologically or

biochemically.

3


Plants produce as amazing diversity of low molecular

weight compounds.

Of the estimated 400,000 500,000 plant species around

the globe, only a small percentage has been investigated

phytochemically and the fraction subjected to biological or

pharmacological screening is even lower.


The ability to synthesize secondary metabolites has

been selected through the course of evolution in different

plant lineage when such compounds address specific

needs.

Floral scent volatiles and pigments have evolved to attract insect

pollinators and thus enhance fertilization.

To synthesize toxic chemical has evolved to ward off pathogens

and herbivores or to suppress the growth of neighboring plants.

4


Chemicals found in fruits prevent spoilage and act as signals (in

the form of color, aroma, and flavor) of the presence of potential

rewards (sugars, vitamins and flavor) for animals that eat the fruit

and thereby help to disperse the seeds.

Other chemicals serve cellular functions that are unique to the

particular plant in which they occur (e.g. resistance to salt or

drought).

Natural Products Drug Discovery and Development

Over the ages, human have relied on nature fro their

basic needs for the production of foodstuffs, shelters,

clothing, means of transportations, fertilizers, flavors and

fragrances, and not least medicine.

Plants have formed the basis of sophisticated traditional

medicine system that have been in existence thousands

of years in countries such as China and India.

5

Natural Products Drug Discovery and Development

About 25% of all prescriptions sold in the US are for

natural products, while another 25% are for structural

modifications of a natural products.

According to Fransworth (1990) claims that 119

characterized drugs are still obtained commercially from

higher plants and that 74% were found from

ethnobotanical information.

. Fransworth, N.R. (1990) In bioactive compounds from plants.

John and Wiley Co..

Primary and Secondary Metabolism

Primary metabolism

The biological reactions are essential to maintain life in living

organisms and are known as primary metabolism.

Plant convert sunlight energy to chemical energy, such as ATP,

NADPH, by the mediation of chlorophyll in chloroplasts and

synthesize sugars and starch from CO2 by using ATP and

NADPH+.

These carbohydrates are stored and used for differentiation

and formation of plant tissues.

6


Secondary metabolism

The metabolisms which are not directly related to maintaining

life, are known as secondary metabolisms.

The products formed by secondary metabolism are called

secondary metabolites.

Secondary metabolite play a role in reinforcement of tissue and

tree body (e.g. cellulose, lignin, suberin), protection against

insects, dieses, and plant regulation (plant hormones).


All organisms need to transform and interconvert a vast number of organic compounds to enable them to live, grow and reproduce.

All organisms need to provide themselves with energy in the form of ATP, and a supply of building blocks to construct their own tissues.

An integrated network of enzyme-mediated and carefully regulated chemical reactions in used for this purpose, collectively referred to as intermediary metabolism, and the pathways involved are termed metabolic pathway.

7


The pathways for generally modifying and synthesizing

carbohydrates, proteins, fats, and nucleic acids are

found to be essentially to same in all organisms, apart

from minor variations.

These processes demonstrate the fundamental unity of all living

matter, and are collectively described as primary metabolism,

with the compounds involved in pathways being termed primary

metabolites.

Primary Metabolisms

Degradation of carbohydrates and sugars generally

proceeds via the well characterized pathways, known as

glycolysis and the kerbs / citricacid / tricarboxylic acid

cycle, which release energy from the organic compounds

by oxidative reactions.

Oxidation of fatty acids from fats by the sequence called

-oxidation also provides energy.

8

Primary Metabolisms

Aerobic organisms are able to optimize these processed by

adding on a further process, oxidative phosphorylation.

This improves the effeiciency of oxidation by incorporating

a more general process applicable to oxidation of a wide

variety of substrates rather then having to provide specific

process for each individual substrate.

Primary Metabolisms

Proteins taken in via the diet provide amino acids, but

the proportions of each will almost certainly vary from the

organisms requirements.

Most organisms can synthesize only a proportion of the

amino acids they actually require for protein synthesis.

Those structures not synthesized, so-called essential

amino acids, must be obtained from external sources.

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Secondary Metabolisms

The compounds which synthesized from the secondary metabolisms are so-called secondary metabolites.

Secondary metabolites are formed in only specific organisms, or groups of organisms, ane are expressioin 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 compounds and their benefit to the organism is not yet known.

It is this area of secondary metabolism that provides most of the pharmacologically active natural products.

Secondary Metabolisms

To make such compounds as sugars, waxes, lignin starch, pigments,

or alkaloids, plants utilize very specific enzymes, each of which

catalyzes a specific metabolic reaction.

The enzymes are proteins called organic catalysts.

These enzymes are coded by specific genes in the plants DNA and are

made via processed we call transcription and traslation.

When there is a series of enzymatically catalyzed reaction in a well-

defined sequence of step, we have what is termed a metabolic pathway.

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Biosynthetic Pathway

Nucleoside diphosphate sugar pathway: cellulose,

hemicellulose, glycosides.

Shikimate-cinnamate pathway: lignin, lignans,

hydrolyzed tannins.

Mixed pathways of 3 and 4: some prenylflavonoids,

quinones and stilbenes.


Primary and secondary metabolites leave a grey area

at the boundary, so that some groups of natural products

could be assigned to either divisions.

Primary metabolites Biochemistry

Secondary metabolites Natural products Chemistry

11

The Classes of Secondary Metabolites

The majority of secondary metabolites belong to one of a number of families, each of which have particular structural characteristics arising from the way in which they are built up in nature (biosynthesis).

The classes of secondary metabolites are: Polyketides and fatty acids

Terpenoids and steroids

Phenylpropanoids

Alkaloids

Others (specialize amino acids and carbohydrates)

Polyketide and Fatty acids

Polyketides are formed by the linear combination of acetate units derived from the building block acetyl co-enzyme A.

The acetate origin of these compounds leads to a preponderance of even-numbered carbon chains.

Many plant oils and animal fats contain long-chain monocarboxylic acids know as fatty acids.

In the fatty acids, the carbonyl group of the acetate units is reduced during the course of the chain assembly process. Dehydrogenation and oxidative processed may subsequently give the unsaturated fatty acids.

12


The common fatty acids have an even number of

carbon atoms, typically C12 C20, linked together in a

straight chain with up four double bonds.

In plants the fatty acids and the corresponding alcohol

are found in leave waxes and seed coating:

Myristic acid (C14) is found in nutmeg seeds.

Palmitic acid (C16) is found in almost all plant oils.

Stearic acid (C18) occurs in long amounts in animal

fat.


Unsaturated fatty acids are important to us in food. Oleic acid is the most widely

distributed, and a major constituent of olive oil.

Linoleic and linolenic acids are most highly unsaturated and are found in linseed oil.

Linolenic acid is easily oxidized by air, and is one of the drying oil used in paint and varnishes.

13


Linolenic acid is oxidized

by plants to jasmonic acid,

which is a signaling

substances that stimulates

plant defense mechanisms.

Arachidonic acid (C20) is a

precursors of the

prostaglandin hormones.


Polyacetylenes

They are a group of naturally

occurring hydrocarbon

derivatives characterized by

one or more acetylenic groups

in their structures.

Araliaceae(),

Campanulaceae(),

Apiaceae(),

Asteraceae, Pittosporaceae(

), and some fungi.

OH

OH

OH

O

O

COOH

OH

OH

Falcarinol

Safynol

Wyerone acid

1,2-dihdroxytrideca-5,7,9,11-tetrayne

15

Terpenes

The terpenes are among the most widespread and

chemically diverse groups of natural products.

Terpenes are a unique group of hydrocarbon-based

natural products whose structures may be derived from

isoprene.

Terpenes are classified by the number of 5-carbon units.

16

Terpenes

Isopentenyl pyrophosphate (IPP) and dimethylallyl

pyrophosphate (DMAPP) combined to yield geranyl

pyrophosphate (GPP), leading to monoterpenes.

Similarly, compounds derived from farnesyl

pyrophosphate (IPP) lead to sesquiterpenes, and

triterpenes are formed from two equivalents of FPP, and

triterpenes are formed from two equivalents of FPP.

Terpenes

The function of terpenes in plants is generally

considered to be both ecological and physiological

Allelophathy

Insecticidal

Insect pollinators

Plant hormone (Abscisic acid, gibberlellin)

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Myrcene

Greek Bay (Laurus nobilis)

Myrcene is found in the essential oil of bay leaves as well as hops. It is used as an intermediate in the manufacture of perfumes.

Hops (Humulus lupulus)

Geraniol and Linalool

OH

Geraniol is isomeric with linalool, constitutes the major part of the oil of roses and is also found in essential oils of citronella, lemon grass, and others.

Geraniol

OH

Linalool

citronella

18

Menthol

OH

Menthol is a well-known monoterpenewhich is found in the essential oil of peppermint and other members of the mint family.

Sesquiterpenoids

More than 10000 sesquiterpenoids have been identified, representing a wide variety compounds of different skeletal types from acyclic to tetra cyclic systems.

-muurolene -cadinene -cadinol-cedrene

logifloene juniperol nootkatin chanootin

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cadinenes

OO

Osantonin

Wormwood(Artemisia maritima)

caryophyllene

Cloves (Syzygium aromaticum) OH

OO

Dandelion (Taraxacum officinale )

tetrahydroridentin B

20

Bioactive Compounds Contributing to Decay Resistance of Taiwania Wood

Fungi and termites are two of the most harmful organisms to wooden structures worldwide

To develop methods for prolonging the service life of wood is one of challenges of wood utilization researchers

As regards decay resistance, Taiwania is classified as the species with an excellent durability in Taiwan

There are several papers dealing with the antifungal and antitermitic activities of Taiwania:

.For. Prod. Industries. 1998, 17, 287.

.Holzforschung. 1999, 53, 487.

.Holzforschung. 2000, 54, 241.

. J. Chem. Eco. 2001, 27, 717.

21

White rot fungi Brown rot fungi Coriolus versicolor Laetiporus sulphureus

Lenzites betulina Antrodia taxa

Pycnoporus coccineus Fomitopsis pinicola

Trichaptum abietinum Phaeolus schweinitzii

Oligoporus lowei

Commonly Occurring Brown-rot and White-rot Fungi

Extractives of Taiwania against White-rot Fungi

Taiwania Heartwood

MeOH extraction

MeOH Extractives

Hex Fr.

Chl. Fr.

EtoAc Fr.

MeOH Fr.

22

Extractives of Taiwania against Brown-rot Fungi

Taiwania Heartwood

MeOH extraction

MeOH Extractives

Hex Fr.Chl. Fr.

EtoAc Fr.

MeOH Fr.

Antifungal Indices of Compounds Isolated from Taiwania Heatwood

0 20 40 60 80 100

L. s.

C. v.

Antifungal index

Suginol

Hinokiol

Ferruginol

-Cedrol

-Cadinol

Helioxanthin

Taiwanin C

Savinin

Taiwanin A

23

Effectiveness of -Cadinol against White-rot and Brown-rot Fungi

Among the compounds isolated from Taiwania heartwood, -cadinolhas demonstrated to possess the highest antifungal effectiveness.

H

OHH

H

OHH

H

OH

24

Quantification of the Cadinane Skeletal Sesquiterpenoids Distribution in Different Parts of Taiwania

GC chromatograms of essential oils distilled from Taiwania heartwood.

H

OH

H

H

OH

H

H

OH 1

2

3

T-cadinol

T-muurolol

-cadinol

The amounts of hexane extractives and essential oils distilled from various parts of Taiwania

0

2

4

6

8

Am

ount

s of

cad

inan

es (m

g/kg

) t-cadinolt-muurolol-cadinoltotal cadinols

Essential oil

Leafs Sapwood Heartwood

n-Hexextracts

Heartwood

Amounts of Cadinanes in Various Parts of Taiwania

6.49

1.77

0.360.04

25

Antifungal Indices of -Cadinol, T-Cadinol, and T-Muurolol

Fungi -Cadinol T-Cadinol T-Muurolol

C. v. 100 47.1 38.8

L. s. 100 100 82

equatorial hydroxyl group

trans ring junction

Diterpenoids

Diterpenoids constitute a major

part of oleoresin.

This group can be divided into

acylic, bicyclic, tricyclic,

tetracyclic, and macrocyclic

structural types.

Diterpenoids are present either

as hydrocarbons or as

derivatives with hydroxyl,

carbonyl, or carboxyl groups.

geranyl-linalool -epimanool

cis-abienol manoyloxide

pimaral

pimarolcembrene

26

Phenolic Constituents

Heartwood and bark

contain a large variety of

complex aromatic

extractives. Most of them

are phenolic compounds,

and many are derived

from the phenylpropanoid

structure.

Classification of Phenolic Compounds

Numbers of C Basic skeleton Name

6 C6 Benzoquinones

10 C6-C4 Naphthoquinones

14 C6-C2-C6 Stilbenes

15 C6-C3-C6 Flavonoids

17 C6-C3-C2-C6 Norlignans

18 (C6-C3)2 Lignans

n (C6-C3-C6)n Condensed tannins

27

Phenolic Compounds

CHO

OCH3OH

Vanillin

OCH3

CH

O

CHH2COH

GLUCOSE

Coniferin

CHO

OCH3OH

CH3O

Syringaldehyde

OHOCH3

Guaiacol

OHOCH3

CH2CHCH2

Eugenol

O

OOCH3CH3O

2,6-Dimethoxybenzoquinone

Summery of the Biogenetic Connection between a Selection of Familiar Phenolic metabolites

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29

LignansIsolation Procedures

Lignans can be isolated from the bark, fruit, heartwood, leaves, roots and resin of plants

Most isolation procedures involve solvent extraction, chromatography separations, and crystallization

Lignan yields can vary from 0-30%

30

LignansCommercialization

Large amount of research devoted to investigating medicinal properties of lignans Particularly from tropical hardwoods and grasses

Example 1999 reference: 35 lignans isolated from the twigs of Tazus mairei

Antiviral Antitumor Biocidal Bioactive Agents

31

The heartwood of Taiwania is

yellowish red with

distinguished purplish pink

streaks, which fascinates

people very much.

The yellowish red color in

Taiwania is susceptible to

change to dull black after

exposure it to nature

environment.

32

Optical Micrographs of Different Parts of Taiwania

Optical Micrograph Observation of Discoloration in Red Taiwania Heartwood

2 weeks 4 weeks

After exposed Taiwania heartwood under indoors condition, its color changed from red to dull black.

33

Color Compounds Isolated from Taiwania Heartwood

O O

O

O

O

O

OH

H

H OH

O

OO

O

OO

OO

O

OO

O

O

O

OO

OH

OH

OOH

O O

OH

OMe

CHO

OH

CHO

Taiwanin A Savinin Helioxanthin Pluviatolide

Taiwanin I Ferruginol T-Cadinol Secoabietane dialdehyde

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Cytotoxicity of Lignans and Sesquiterpenoids from Taiwania Heartwood (ED50 values in mM)

Taiwanin A

Taiwanin E

-CadinolDimethylmatairesinol

Cytotoxicity of Dibenzyl--butyrolactone Type of Lignans Isolated from Taiwania Heartwood

An unsaturated double bond between C7-C8 and/or C7-C8is associated with stronger cytotoxicity.

The presence of two 3, 4-dimethoxyphenyl groups in lignans may increase the cytotoxicity, as dimethyl-matairesinol was stronger than arctigenin and hinokinin

O

O O

OO O

OO

O

OO

O

O

R

R

RR

35


An unsaturated double bondbetween C7-C8 and/or C7-C8is associated with stronger cytotoxicity.


O

O O

OO O

OO

O

OO

O

O

R

R

RR


An unsaturated double bondbetween C7-C8 and/or C7-C8is associated with stronger cytotoxicity.


O

O O

OO O

OO

O

OO

O

O

OMeMeO

MeO

MeO

36

Cytotoxicity of Arylnaphthalide Type of Lignans Isolated from Taiwania Heartwood

Taiwanin E showed the strongest cytotoxicity in this group.

It appears that the hydroxyl group at the C7 position enhances the cytotoxicity.

OO

O

OO

O

OO

O

OO

O

OH

O

OO

OO

O

Cytotoxicity of Arylnaphthalide Type of Lignans Isolated from Taiwania Heartwood

Taiwanin E showed the strongest cytotoxicity in this group.

It appears that the hydroxyl group at the C7 position enhances the cytotoxicity.

OO

O

OO

O

OO

O

OO

O

OH

O

OO

OO

O

37

Flavonoids/TanninsIsolation of Flavonoids

Isolation of flavonoids accomplished through solvent extraction Hot water Alcohols

Solvent fractionation of extract Salting out Crystallization

Flavonoids/TanninsIsolation of Flavonoids

Flavonoids concentrated in certain parts of plants

Plant sources Pulp of fruits

Broccoli, green peppers, onions, etc.

Green tea, red wine

Herbs

Tree bark

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Blockade of tumor induction by EGCG

O

OC

O

OH

OH

OH

OH

OH

OH

OH

OH

(-)-epigallocatechin-3-gallate

GST: glutathione S-transferaseGlcT: UDP-glucuronyl transferaseROS: reactive oxygen

(superoxide anion, hydrogen peroxide,hydroxyl radical, nitric oxide, peroxynitrite, and nitric dioxide anion)

RH: procarcinogens(aflatoxins, poly-cyclic aromatic hydrocarbons, and nitrosamines)

Chemical Composition of Tea

Caffein: 3-4 %

Catechins (including Tannin): 15-30 %

Flavonols and Metal: 5 %

P,K, Ca, Mg, Mn, Zn, Cu, Al

Vitamins: A, B1, B2, C, E, Nicotinic acid

39

Flavonoids/TanninsPolymerization Reactions

Condensed tannins are formed through the polymerization of flavan-3-ol (catechin) and flavan3,4-diols (leucoanthocyanidins) In tree, polymerization

through acidic enzymatic non-oxidative coupling

2-50 units Typically 2-8

Linkages can be through a variety of sites

O

OHOH

Flavan-3,4-diols(Leucoanthocyanidins)

O

OH

Flavan-3-ols(Catechins)


Examples of linkages 4 alpha 8 4 beta 8 4 beta 6

Stereochemistry A variety of monomers

O

A

B

C2

3

456

78

2'3'

4'

5'6'

40


O

OH

OHOH

OH OH

HO

O

OH

OHOH

OH

HO

O

OH

OHOH

OH

HOO

OH

OHOH

OH

O

O

OH

OHOH

OH

HO

O

OH

OHOH

OH

OH

OHOH

OH

O

HO

HO

(H+)

-H2O

+ H+

(+)

- H+


Flavan-3-ols are incapable of polymerizing without flavan 3,4-diol

Flavan-3-ol form terminating unit

O

OH

O OH

OH

OH

OH

OH

O OH

OHOH

HO HO

HO

HO

41


All 4-8 linkages

This structure has

been isolated from

pine bark

O

OH

OHOH

OH

OH

OHOH

OH

O

OH

OHOH

OH

O

HO

HO

HO


Flavan-3-ols can polymerize through an oxidative enzymatic coupling reaction forming oligo and polymeric materials Linkages 8-6 Causes loss of

brightness in wood

O

OHOH

OH O

OOH

OHOH

O

HO

HO

42

Flavonoids/TanninsLocation in Tree: Tannins

Western hemlock Bark: 18% tannins Wood: 2% tannins

Quebracho In wood, tannins found in heartwood Majority (80%) located in vessel lumina Tannins deposit in dead cell starting at CML and

ending in secondary wall

43

Condensed TanninsProperties

The term condensed tannins refers to a mixture of polyflavonoids of different MW (500-5000) characterized by different linkages, functional groups, and stereochemistry.

Protein binding capacity: tannins will bind with proteins causing them to precipitate. This was the definition of tannins: compound which

will precipitate proteins.

Condensed TanninsSources

Condensed tannins more prevalent in

hardwoods but present in softwoods

Wattle (Acacia - Southern Africa)

Quebracho (Schnopsis - South America)

Mangrove (Rhizophora -)

Hemlock (North America)

44

Condensed TanninsBiological Significance Insects/Animals

Protection of plants against insects/animals Some evidence for/some against

Bad Taste/Astringency (bitter taste) Appears to be major factor

Particularly bad for insects not used to tannins

Animals: tannins reduce digestion of food Interaction with digestion enzymes

Toxic to bacteria

Condensed TanninsBiological Significance - Fungus

Pine calluses: created by fungal invasion

Tree forms calluses as protective tissue

Calluses contain high levels of tannins (Chinese 50-80%)

Concentrations of tannins as low as 0.1% or 0.8 % have been shown to retard the growth of a large number of parasitic fungi

Quote: Edwin Haslam (tannin chemist)

serious and nagging fear that a part at least of (their) scientific career(s) has been spent inspecting the loot in the garbage bin of plant metabolism

45

Hydrolyzable TanninsStructure

Polymers of a sugar (usually glucose) with one or more polyphenolic carboxylic acids: linked through ester linkages

Gallotannins: Gallic acid polymer Ellagitannins: Ellagic Acid polymer

COOH

OHOHHO

Gallic Acid

OHOH

CO

OH

OHC

HO

O

HOO

Digallic Acid

OH

C

O

OHOH

O

C

HO

O

O

Ellagic Acid

Hydrolyzable TanninsPolymer Structure Example

O

OO

OH

O

O

HO

HO

OH

O

HO

HO

O

OHOHO

OHOH

OH

O

OH

OH

OH

O

O

OH

OHO

O n

Sugar

46

Hydrolyzable TanninsTree Information

Rare to nonexistent in softwoods

Hardwoods which contain large amounts:

Oak (gallic and ellagic tannins)

Eucalyptus (Ellagitannins)

Chestnut (gallic tannins)

Myrobalan fruits (cherry plum)

Hydrolyzable tannins located in heartwood

Condensed TanninsUses

Leather tanning: 10,000+ year old industry Vegetable tannins & chrome Tannins interacting with proteins in hides

Adhesives In phenol formaldehyde systems, tannins

speed up the set:

Oil well drilling fluids: old but effective use: taken over by chrome lignosulfonates

47

Stilbene -Diphenylethylene

Alkaloids

Derived from plants

With a basic character (hence the term

alkaloid from alkali)

Contained a nitrogen based heterocylic

ring within their molecules

48

Alkaloids can be sub-categorized according to: Monocyclic

alkaloids Bicyclic alkaloids Polycyclic alkaloids

49

OpiumPapaver somniferum - used for 5000 years. Arabs introduced opium to China in 7th century. Opium addiction became a problem so Chinese officials outlawed it. England traded opium to China for goods even though it was illegal in their own country and in China. China and England fought two wars over the English import of opium, China lost both, ceded Hong Kong to British at end of first war. Use of opium in China did not drop until Communist Revolution in 1949. Most opium is currently grown in SE Asia.

Morphine is purified from opium and is a very potent painkiller.

Narcotic analgesics

OR

OH

N CH3

O

R = H morphineR = CH3 codeine

Ephedra sinica,

Ephedra intermedia,

Ephedra equisetina

Ma huang

Ma Huang or Ephedra has been used in China since 2800 BC It was used primarily for colds, asthma, hayfever, bronchitis, edema, arthritis, fever, hypotension and hives. Potency is based upon the herb's alkaloid content. Side effects include increased blood pressure, heart rate and anxiety. FDA suggest that those with heart (disease or high blood pressure), thyroid, diabetes or prostrate problems may be affected adversely. It should definitely NOT be taken with antihypertensive or antidepressant drugs.

CCCH3

NH

CH3HH OH C

CCH3

NH

CH3HOH H

d-pseudoephedrinel-ephedrine

50

Vinblastine is a drug used in the treatment of cancer. It interferes with the multiplication of cancer cells and slows or stops their growth and spread in the body.

Catharanthus roseus

Berberis fremontii

secondary metabolites from plants plant secondary metabolites

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