Lectures in Natural Products Chemistry

MSc. Zeid A. N.

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University of Technology Applied Sciences Department School of Biochemical Technology 3rd Stage

Natural Products Chemistry

By MSc. Zeid Abdul-Majied Nima

Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Syllabus
Topics
Introduction Brief History

Page

Major Groups of antimicrobial compounds from plants

Natural Products Compounds Phenolics and Polyphenols Terpenoids and essential oils Alkaloids Lectins and Polypeptides Basic Macrolides antibiotics Nucleoside peptide antibiotics Bleomycin group antibiotics Penicillins Cephalosporins Streptomycin group antibiotics Neomycin Gentamicin Chloroamphenicol Efficacy Identification of Natural Organic Compounds Infra red Spectrometry (IR- Spectrometry Ultraviolet-Visible Spectrometry (UV-visible Spectrometry) Nuclear Magnetic Resonance (1H NMR)

Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Introduction *Finding healing powers an ancient idea (Ethnomedicine, ethnobotany, ethnopharmacology. etc.) Which utilizes plants and animals products to maintain peoples health. *There is evidences that Neanderthals living 60,000 years ago in present Iraq used plants such as hollyhock. *Currently , of the one quarter to one-half of all pharmaceuticals ,having higher plant origins . *Most antimicrobials having bacterial and fungal origins. *Tow reasons to be interested in antimicrobial plant extract : 1. The life span of any antibiotics is limited. 2. Overprescription and misuse of traditional antibiotics. Brief History *Arab world continued to build their medicine. *Asian Cultures was also busy compiling their own pharmacopoeia (Note: three type , British pharmacopoeia (BP), US pharmacopeias (USP) , European Pharmacopoeia ). * In North America : 1) Indigenous cultures (Native Americans) 2) Americans from European origins * Mainstream medicine is increasingly receptive to the use of antimicrobial and other drugs derived from plants (products of microorganisms or Synthized derivatives). Major Groups of antimicrobial compounds from plants Plants have an almost limitless ability to synthesize aromatic substances , most of which are phenols or their oxygen-substituted derivatives. Most are secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be less than 10% of the total. In many cases, these substances serve as plant defense mechanisms against predation by microorganisms, insects, and herbivores. Some such as terpenoids, give plants their odors; others (quinones and tannins ) are responsible for plant pigment. Many compounds are responsible for plant flavor (e.g., the terpenoid capsaicin from chili papers ), and some of the same herbs and spices used by humans to season food yield useful medicinal compounds.

Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Natural Products Compounds 1- Phenols and Polyphenols (a) Simple phenols and Polyphenols

Compounds name Cinnamic acid

Chemical Structure
CH HC C OH O

Antimicrobial or pharmacological effects Viruses, Bacteria, Fungi

HO

CH HC HO COOH

Caffeic acid

Viruses, Bacteria, Fungi

HO

Catechol
HO
HO

Toxic to Microorganisms

Pyrogallol

HO

Toxic to Microorganisms
HO

HO

Eugenol
H 3 CO

CH 2

general

Mechanisms : enzyme inhibition by the oxidized compounds through reaction with sulfhydral groups or through more non-specific intractions with the proteins. (b) Quinones : are aromatic rings with two ketone substitution.

Lectures in Natural Products Chemistry

O OH

MSc. Zeid A. N.

OH

[ 1 ,4 ] B e n z o q u i n o n e

B e n z e n e - 1 ,4 - d i o l

Diketone ( Quinone )

Diphenol (Hydroquinone)

Compounds name Quinone

Chemical Structure
O O

Antimicrobial or pharmacological effects General antimicrobial

CH
3

O H 3 CO

CH

Ubiquinone (Coenzyme Qn)

H 3 CO O
O

General antimicrobial
CH
3

Naphthaquinone (Vitamin K)
O
OH O OH

Anti hemorrhagic activity related to its oxidation in body tissue

HO

OH OH

Hypericin

HO

Antidepressant and General antimicrobial

OH

OH

Mechanisms : Quinones are known to complex irreversibly with nucleophilic amino acid in proteins leading to inactivation of protein and loss of function. Targets in the microbial cell are surface exposed adhesions ,cell wall polypeptides, membrane-bound enzymes. (c) : Flavonoids Flavone : phenolic structure containing one carbonyl group Flavonol : Flavone + 3-OH groups Flavonoid : Flavonol + C6-C3 unit linked to an aromatic ring Compounds name Chemical Structure Antimicrobial or pharmacological effects 5

Lectures in Natural Products Chemistry

OH

MSc. Zeid A. N.

HO

O OH

Catechins
OH OH

Vibro Cholera Streptococcus mutans Shigella

HO

Chrysin

HIV and RSV (Respiratory Syncytial Virus)

OH

O
OH

HO

O OH

Quercetin

HIV and RSV

OH

O OH O CH 3

Hespertin

HO

HSV , poliovirus type1 and parainfluenza type3

OH
O

Isoflavone
O

microorganisms
HO OH OH

Phloretin
OH O

microorganisms

HO

Galangin
OH OH O

Gram-positive bacteria , fungi, viruses (HSV-1 and Coxsackie virus type 1)

Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Glycyrrhizin

HIV

swertifrancheside

HIV

Mechanisms: antimicrobial activity is due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls. d-Tannins: General name for a group of polymeric phenolic substances capable of tanning leather or precipitating gelatin from solution (astringency) their MW range 500-30,000 ,found in bark , wood, leaves, fruits and roots. -Tannins : a) Hydrolyzable tannins (esters of gallic acid +D-glucose) b) Condensed tannins (flavonoid monomers) -Biological activity: a) stimulation of phagocyte cell b) host-mediated tumor activity c) wide range of anti-infective actions -Tannins as antimicrobial agent : toxic to filamentous fungi , yeasts, and bacteria. Compounds name Chemical Structure Antimicrobial or pharmacological effects

Pentagalloylglucose (hydrolysable tannin)

Lectures in Natural Products Chemistry

MSc. Zeid A. N.

proanthocyanidin (condensed tannin)

Mechanisms: Complex with non-specific forces such as hydrogen bond and hydrophobic effects ,as well as, covalent bonds formation also complex with polysaccharides. e- Coumarins: are phenolic substances made of fused benzene ring and !-pyrone ring. -Coumarins functions : a) antithrombolic b) anti-inflammatory c) Vasodilatory activities Compounds name coumarin
O O

Chemical Structure
O O

Antimicrobial or pharmacological effects Candida albicans

CH 3

warfarin
OH O

Highly toxic : Intraction with eukaryotic DNA (human)

Hydroxycinnamic acid

Inhibits Gram-psitive bacteria

Phytoalexins (ficusin)

Antifungal activity

2-Terpinoids and Essential oils -the fragrance of plants is carried in the so called "quanta essentia" or essential oil fraction. 8

Lectures in Natural Products Chemistry

MSc. Zeid A. N.

-based on Isoprene structure :

CH 3 CH2 H 2C

isoprene

-Terpenes and Terpenoids: Terpenes : are isoprene derivatives their chemical structure C10H16 (terpene) monoterpene ,diterpene , triterpene and tetraterpene. Hemiterpene (C5H6 ), Sesquiterpene (C15 ). Terpenoids: are terpenes containing oxygen. -In general all terpenoids active against bacteria, viruses , fungi and protozoa. Compounds name Antimicrobial or pharmacological effects

Chemical Structure
C H3

Menthol (mono)
H 3C CH 3

OH

Camphor (mono)

Farnesol (sesquiterpenoid)

Cerebral malaria

Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Artemisin (sesqui)

antimicrobial

Betulinic acid (triterpenoid)

Anti HIV

O H3 CO CH3 N H CH3 HO

capsaicin

Biological active in human : affecting the nervous , cardiovascular and digestive system Uses: analgesic and antimicrobial

aframodial

A broad spectrum antimicrobial

petalostemumol

Bacillus subtills and streptococcus aureus

Mechanisms: the mechanisms of action of terpenes are speculated to involve membrane disruption by the lipophilic compounds.

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Lectures in Natural Products Chemistry

MSc. Zeid A. N.

-Alkaloids: Heterocyclic nitrogen compounds -up to present time , about 800 kind of indole alkaloids have been isolated . -Very active compound such as : 1- Reserpine : an antihypertensive 2- Ajmaline : an anti cardiac arrhythmic 3- Vincristine : an antileukemic , the finding ofantileukemic alkaloids !vincristine" from catharanthus roseus (vinca rosea) confirmed the importance of indole alkaloids as objects of pharmaceutical studies. -Most of the plants which contain indole alkaloids grow in the tropical regions of the world .

Compounds name

Chemical Structure

Antimicrobial or pharmacological effects

Anesthesia (The principal Morphine

alkaloid in opium and the prototype opiate analgesic and narcotic. Morphine has widespread effects in the central nervous system and on smooth muscle.)

Anesthesia (An opioid Codeine (methymorphine)

analgesic related to MORPHINE but with less potent analgesic properties and mild sedative effects. It also acts centrally to suppress cough.)

Heroin (diacetylmorphine)

Anesthesia( A narcotic
analgesic that may be habitforming)

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Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Solamargine

Against HIV infections and intestinal infections associated with AIDS

Berberine

Trypanosomes (Trypanosomes are a group of kinetoplastid protozoa distinguished by having only a single flagellum) and plasmodia (Plasmodium is a genus of parasitic protozoa. Infection with this genus is known as malaria.)

Mechanisms: (of action as antimicrobial ) is attributed to their ability to intercalate with DNA and /or cell membraine. -Lectins and Polypeptide - lectin : (phytoagglutinin) Mwt 17,000-400,000 -A group of proteins , widely distributed in nature that have the ability to agglutinate erythrocytes and many other types of cells. -Found primary in seeds of plants , but also occur in roots, leaves and bark. -found in plants , invertebrates and several vertebrates . -The term !Phytoagglutinins" is used to refer to plant lectins. -Lectin exhibit a variety of un usual biological properties : 1- Reaction with human blood groups 2-Induce mitosis in lymphocytes 3-some plant lectin mimic the direct effects of insulin Examples : -Soybean agglutinin and Concanavalin A : shown to agglutinate cell lines transformed by viral or chemical carcinogens. -Soybean agglutinin : used in bone marrow transplants in patients with severe combined immunodeficiency. 12

Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Polypeptides : antimicrobial peptide were first reported in 1942 , that positively charged and contain disulfide bounds . -thionin : anti yeast . G+ , GThionin AX! And AX2: antifungi Fabatin E.coli Mechanisms: formation of ion channel in the microbial membrane or competitive inhibition of adhesion of microbial proteins to host polysaccharide receptors. -Basic Macrolides Antibiotics Macrolide : is a group of natural antibiotic possess lactone ring and one to three sugar moieties (macrocyclic lactone) Uses: chemotherapeutic agents active against Gram-positive bacteria. Classification: classified according to the ring size of the lactone moiety ; 12- ,14- ,and 16membered rings. General Source: streptomyces species Mechanisms: the macrolide prevent protein synthesis by arresting the translocation movements that permit the sequential linking of amino acids to produce the ribosomal protein. Examples: 1- Erythromycin and Oleandomycin (14-membered) 2- Leucomycin, Spiramycin and Platenomycin (16-membred) 3- Medicamycin (12-membered) Compounds name Chemical Structure Antimicrobial or pharmacological effects
A bacteriostatic antibiotic macrolide produced by Streptomyces erythreus. Erythromycin A is considered its major active component. In sensitive organisms, it inhibits protein synthesis by binding to 50S ribosomal subunits. This binding process inhibits peptidyl transferase activity and interferes with translocation of amino acids during translation and assembly of proteins.

Erythromycin (14-membred)

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Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Leucomycin (16-member)

A macrolide antibiotic produced by Streptomyces kitasatoensis. The drug has antimicrobial activity against a wide spectrum of pathogens.

Platenomycin

(16- member)

Spiramycin (16-member)

A macrolide antibiotic produced by Streptomyces ambofaciens. The drug is effective against gram-positive aerobic pathogens, N. gonorrhoeae, and staphylococci. It is used to treat infections caused by bacteria and Toxoplasma gondii

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Lectures in Natural Products Chemistry

MSc. Zeid A. N.

-Nucleoside peptide Antibiotics Polyoxins : a hybrid nucleoside and peptide structure (5-substituted Uracil nucleoside + peptide) Uses: Agricultural fungicides Classification : A-M polyoxins Source : streptomyces cacaoi Mechanism : inhibits of fungal cell wall chitin biosynthesis , this was predicted when the structure was elucidated . The gross structure of polyoxin mimics that UDP-Nacetylglucoseamine, a substrate of chitin synthetase. Subsequently, the polyoxins are competitive inhibitors for chitin synthetase in fungi, yeasts and insects. General Structure:

O R1 NH

O C HN

COR 2 CH O H H

three amino acid peptide

H OH OH

Examples : Compounds name Chemical Structure Antimicrobial or pharmacological effects

Polyoxin A

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Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Polyoxin B

Polyoxin L

-Bleomycin Group Antibiotics Bleomycin :a group of antitumor glycopeptides antibiotics produced by stryptomyces verticillus. Uses: 1- sequamous cell carcinoma 2- malignant lymphoma 3- antibacterial Classification : BMLs Classification to , A1 , A2, A5, A6 and B1,B2 , B4 Sources: stryptomyces verticillus Mechanisms : prevent protein synthesis in the membrane. A complex of related glycopeptide antibiotics from Streptomyces verticillus consisting of bleomycin A2 and B2. It inhibits DNA metabolism and is used as an antineoplastic, especially for solid tumors.

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Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Examples : Compounds name Chemical Structure Antimicrobial or pharmacological effects

Bleomycin A2

Bleomycin B2

The Penicillins In 1928 Alexander Fleming , found that the plate culture of staphylococci in his laboratory become contaminated with spores of common green mould ,Penicillinium notatum .He noted the bacterial colonies in the immediate neighborhood of spores were showing signs of lysis. Penicillin as active has become called until after 1940 when Flory and Chain and their colleagues isolate it and described its properties in details. Uses: Gram-positive bacteria Source: P.notatum , P.chrysogenum

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Lectures in Natural Products Chemistry

MSc. Zeid A. N.

General Structure:

H N

CH 3

N O COOH
Examples: Compounds name

CH 3

Chemical Structure

Antimicrobial or pharmacological effects

A penicillin derivative commonly used in the form of its sodium or potassium salts in the treatment of a variety of infections. It is effective against most gram-positive bacteria and against gramnegative cocci. It has also been used as an experimental convulsant because of its actions on GAMMAAMINOBUTYRIC ACID mediated synaptic transmission.

Penicillin G

Penicillin V

A broad-spectrum penicillin antibiotic used orally in the treatment of mild to moderate infections by susceptible grampositive organisms.

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Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Amoxycillin

A broad-spectrum semisynthetic antibiotic similar to AMPICILLIN except that its resistance to gastric acid permits higher serum levels with oral administration.

Ampicillin

Semi-synthetic derivative of penicillin that functions as an orally active broad-spectrum antibiotic

Cephalosporins Source: cephalosporum genus (muld) Uses: Gram-positive bacteria and Gram-negative bacteria General Structure:

R1

NH

N O COOH CH 2R 2

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Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Examples: Compounds name Chemical Structure

O H 2C C NH

Antimicrobial or pharmacological effects

Cephaloridine (Keflodin)

S N O COOH O HC C NH CH 2 N

Cephalexin (Keflex)

NH 2 N O COOH CH 2 CH 3

Streptomycin group antibiotics Source: streptomyces griseus Uses: Gram-negative bacteria Example : Neomycin Compounds name Chemical Structure Antimicrobial or pharmacological effects

Neomycin

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Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Gentamicin Source : Micromonospora purpurea Uses : Gram-positive bacteria Structure :

Tetracyclines Sources: streptomyces aureofaciens and S. rimosus Uses: most active against gram-positive bacteria General Structure

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Lectures in Natural Products Chemistry

MSc. Zeid A. N.

Efficacy Invitro experiments : i. Bacteria and fungi : Initial screening for antibacterial and antifungal compounds may be performed with pure or crude extracts. The tow most commonly used screens are : 1)The broth diffusion assay, 2) Disc diffusion assay " zone inhibition assay". In addition to these assays , antifungal phytochemicals can be analyzed by : Spore germination assay , usually 25o for 24 h. Of curse, after initial screening more detailed studies should be conducted, At this stage, more specific media can be used and MIC (MIC : the minimum concentration of antibiotic that kills 99.9% from the microorganisms) can be effectively compared to those of a wider range of currently used antibiotics . Viruses :Invitro antiviral screening assays Determination of viral infectivity under tow conditions : 1) In Culture cells during virus multiplication in the presence of a single compound (A-S) or a mixture of compounds , e.g. plant extracts (A-M) 2) After extracellular incubation with a single compound (V-S) or a mixture of compounds (V-M). Assays: 1) Plaque inhibition assay : (only for viruses which form plaques in suitable cell system) Titer determination of a limited number of viruses in the presence of a nontoxic dose of the test substance. Application : A-S 2) Plaque inhibition assay : (only for viruses which form plaques in suitable cell system) Titer determination of residual virus infectivity after extracellular action of test substance(s) . Application : V-S, V-M 3) Inhibition of virus-induced cytopathic effect (for viruses that induce cytopathic effect but don#t readily form plaques in cell cultures) Determination of virus-induced cytopathic effect in cell monolayers cultured in liquid medium, infected with a limited dose of virus, and treated with nontoxic dose of the test substance(s). Application : A-S, A-M 4)Virus yield reduction assay Determination of the virus yield in tissue cultures infected with a given amount of virus and treated with a nontoxic dose of the test substance(s). Virus titer determination carried out after virus multiplication by the plaque test or the 50% tissue culture infective dose end-point test . Application : A-S, A-M 5) End-point titer determination technique Determination of virus titer reduction in the presence of twofold dilution of test compound(s). This method especially designed for antiviral screening of crude extracts. Application : A-S, A-M 6) Assays based on measurement of specialized functions and viral products (for viruses that don#t induce cytopathic effects or form plaques in cell cultures) Determination of virus specific parameters, e.g. inhibition of cell transformation, immunological tests detecting antiviral antigens in cell cultures (HIV, cytomegalovirus). Application : A-S, A-M, V-S, V-M 22 ii.

Lectures in Natural Products Chemistry

MSc. Zeid A. N.

iii.

Protozoa and helminthes : more complicated than screening for activity against bacteria, fungi or viruses. Tow methods to assay compounds: 1) 66-h tissue cultures of the protozoa were exposed to various concentrations of extracts and then the MIC were determined. 2) The fluorescence assay ( radiolabel microorganisms) of protozoa viability in microtiter wells.

In vivo experiments Tow prime examples of animal studies from the recent literature are the descriptions of the effect of extracts on rats and mice.

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