6.Aptosimum Literature Chapter 2

Chapter 2

Phytochemistry of Aptosimum procumbens

2.1 Introduction

Aptosimum procumbens Burch (= A. depressum) of the tribe Aptosimae belongs to the Scropulariaceae sensu stricto family of the order Lamiales.1 Scrophulariaceae is one of the largest plant families and is comprised of about 190 genera and 4000 species. Plants from this family are mostly woody herbaceous shrubs and are found predominantly in the temperate regions of the world.2 They are distinguished from related families with relative ease, but many plants are assigned to this family because they lack distinguishing characteristics that would place them in the other specific families. Therefore, Scrophs share some of the characteristics of plants of related families and this may negate the possibility that Scrophulariaceae is a distinct clade. As a result of this, there are doubts as to whether the family is monophyletic or should rather be classified as polyphyletic or paraphyletic.1 Phytochemical investigations can provide a valuable input with regards to the chemotaxanomical studies of this family.

A. procumbens (Fig. 1) is mainly referred to as “carpet flower” but shares a variety of vernacular names such as “brandbossie/blare”, “Karoo violet/flower” and “kankerbos” with other related species of Aptosimum. A common name is given to a plant based on certain characteristics of the plant. A. procumbens is a prostrate mat-forming species hence the name “carpet flower”. The plant has strong woody procumbent stems with short lateral, dense leafy and floriferous branches. Another characteristic feature is the violet trumpet-like flowers that bloom in the summer or after rainfall (hence the name “Karoo violet”). The dwarf shrub grows mostly in dry soil, on rocky terrain in full sun, in flood plains and is found in semi-arid regions of southern Africa (commonly in the Karoo region).3,4

http://www.museums.org.za/bio/plant/scrophulariaceae/aptosimum_procumbens.htm Figure 1: A. procumbens

Aptosimum has been used as a traditional medicine in South Africa3,4 and in parts of Europe, such as Germany for the treatment of a variety of ailments such as water retention and micturition difficulties, diptheria, ringworm, impetigo lesions and krimpsiekte in sheep.5 It may be administered as a tea, gargle, decoction or paste depending on the disease it is being used to treat. When applied to the skin, the foliage may cause a blister suggestive of a burn and it is, therefore, also referred to as brandbossie. 6

A variety of Aptosimum species such as A. indivisum, A. decumbens, A. albomarginatum, A. angustifolium, A. spinescens, A. glandulosum, A. arenarium, A. sp, A. lineare and A. steingroeveri with similar physical and medicinal characteristics exist.4,6 In general, most of the species are used in wound healing or for the treatment of gastrointestinal disturbances. A. indivisum and A. sp are also referred to as the Karoo violet.3 A. sp is an unidentified species that has been used for snakebites and epilepsy. 7 A. albomarginatum is an abortifacient and is used to assist in the expulsion of the placenta and regulates the menstral cycle. A. spinescens also called “kankerbos”, has been used as treatment for cancer, hypertension and haemorrhoids and the leaves of A. decumbens may be chewed to improve memory.6

Schrophulariaceae is a rich source of iridoid glycosides, other compounds sometimes found in this family include orobanchins, triterpenoid saponins, cardiotonic glycosides and flavonoids.8 Plants containing iridoids generally have anti-inflammatory properties.9 Some of these plants, such as Devil’s claw (Harpagophytum procumbens), are used as traditional medicines10.

7 To date only two species of Aptosimum have been investigated namely, A. spinescens and A. indivisum. Eight lignans: sesamin (2.1), spinescin (2.2), piperitol (2.3), pinoresinol (2.4), pinoresinoldimethylether, pinoresinolmonomethylether, aptosimon (2.5) and aptosimol (2.6), have been isolated from A. spinescens.11 In a paper on the phytochemistry of A. indivisum the plant was wrongly identified as Craterocapsa tarsodes (Campanulaceae) (FR van Heerden, personal communication). Shanzhiside methyl ester (an iridoid) (2.7), verbascoside (2.8) and the flavanone pinocembrin 7-O-b- neohesperidoside (2.9) were isolated from A. indivisum.12 A. procumbens has been used as a traditional medicine in the Cape and the Karoo regions. In our laboratories the crude extract of A. procumbens showed moderate anticancer activity against three cell lines and we decided to perform a phytochemical investigation on the plant.

O Ar1 1 2 2.1 Ar = Ar = 3,4-methylendioxyphenyl 2.2 Ar1 = 3,4 methylendioxyphenyl Ar2 = 3,4-dimethoxyphenyl Ar 2 O 2.3 Ar1 = 3,4-methylendioxyphenyl Ar2 = 3-methoxy-4-hydroxyphenyl

2.4 Ar1 = Ar2 = 3-methoxy-4-hydroxyphenyl

O R1

R2 2.5 Ar = 3,4 methylendioxyphenyl R1, R2 = O

2.6 Ar = 3,4-methylendioxyphenyl Ar Ar O R1= OH R2= H

CO Me HO 2 H

H HO OH O HO O HO

HO 2.7

8 O

HO HO O O O OH O

HO OH H3C O HO OH

HO OH 2.8

HO HO O O O HO

H3C O HO OH O HO OH 2.9

2.2 Literature Review on Iridoids

2.2.1 Structural Classification

Iridoids are monoterpenes and are found as natural constituents in a large number of plant families. These compounds have a characteristic cyclopenta[c]pyranoid skeleton also known as an iridane skeleton (cis-2-oxabicyclo [4, 3, 0 ]nonane) (2.10). They occur mainly in the form of glycosides. A methyl group (C-10) is found most commonly at the C-8 position and is rarely absent.13 The term iridoid was derived from the name s iridomyrecin, iridolacton and iridodial, compounds that are present in the defense secretions of certain ant species of the Iridomyrmex genus.14

6 4 5 3 7 O 8 9 1

2.10

9 There are four main categories of iridoids: agylcone iridoids, secoiridoids, bisiridoids and iridoid glycosides (the most abundant).13 Iridoids generally have nine carbons with a tenth carbon often bonded to C-4. This carbon may be a methyl group or it may form part of a carbonyl or secondary alcohol functional group. Structural variations and diversification of iridoid types are achieved by the introduction of additional carbons, functional groups and double bonds into the skeleton.13

Simple aglycone structures such as nepetalactone (2.11) are found in plants. This compound was isolated from Nepeta cataria (Lamiaceae), otherwise known as catnip, and is an (sometimes infamous) attractant of cats.15 However, most non-glycosidic iridoids form part of modified structures such as alkaloids, polycyclic compounds, polyesters and intramolecular ethers.13

A large portion of iridoid glycosides can be characterized as glucosides with a glycosidic linkage between the anomeric hydroxyl of D-glucose and the aglycone C-1 hydroxyl. A simple example of this is loganin (2.12), the biosynthetic precursor of many iridoids.16 In some rare cases, the glycosidic linkage may occur between the C-11 hydroxyl of the aglycone and anomeric carbon hydroxyl of D-glucose, as in ebuloside (2.13) isolated from the Caprifoliaceae family.13 The sugar type and complexity may also vary, for example catalpol (2.14) may have an additional rhamnosyl attached at C-6 to form 6-O- a-L-rhamnosylcatalpol (2.15). This is also an example of an iridoid that lacks an tenth carbon.17

CO2CH3 H H

HO O O

H H H 3C H3C O O-Glc

2.11 2.12 O-Glc

RO H

O O O O

H H C 3 O OGlc OH 2.14 R = OH O 2.13 2.15 R = Rhamnosyl

10 Secoiridoids arise as a result of the cleavage of the 7,8 -bond of the cyclopentane ring. Secologanin (2.16) is a secoiridoid with a vinyl group at C-9. Various other structural modifications give rise to different secoiridoids. Aglycone secoiridoids do exist but are a rarity.18

O CO 2CH3

O-Glc 2.16

Bisiridoids form as a result of dimerization of both iridoids and secoiridoids. Picconioside I (2.17) is a bisiridoid that consists loganin (part a) esterified with deoxyloganin (part b).19 Other structural variations include modification of the iridane skeleton and it’s functional groups. The methyl group (C-10) may be oxidized, giving rise to a secondary alcohol as in catapol (2.14). Epoxidation of the cyclopentane ring as in catapol (2.14) is another example of ring modification. Acetoxy groups may also be introduced as in lamioside (2.18). In deutzioside (2.19) the methyl group (C-10) is absent.13

CO2CH3

O C O HO HO OH H

OGlc

O O O part a O AcO H H CH OGlc 3 O-Glc O-Glc

part b 2.17 2.18 2.19

The presence of unsaturation as in aucubin (2.20) or monotropein (2.21) also leads to differentiation. The introduction of a hydroxyl at, or oxidation of C -6, C-7 and C-8 as in verbenalin (2.22), loganin (2.12) or monotropein (2.21) results in further structural diversification.

11 A notable structural variation is that certain iridoids possess carboxyl groups (C-11) and others do not. Iridoids can, therefore, also be classified as carboxylated and non- carboxylated. Moreover, they are derived from different biosynthetic precursors.20

COOH CO 2CH3 HO O H H H

O O O HO

H H H OH HO O-Glc O-Glc O-Glc 2.20 2.21 2.22

2.2.2 Biosynthesis of Iridoids

The basic building blocks of terpenoids are C-5 isoprene units, in the form of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Iridoids are C10 monoterpenes and are derived from geraniol.21 The isoprene units can be identified in most terpenoids with relative ease, as is evident in the iridane skeleton (2.23). Carboxylated (C-11) and non-carboxylated iridoids are formed from different basic iridoid precursors by two different routes. Loganin (2.12) and 8-epi-loganin (2.24) are key intermediates in the metabolic pathway.20

CO 2CH3 H

HO O O

H H C 3 O-Glucose 2.23 2.24

Isopentenyl pyrophosphate can be formed from mevalonic acid (MVA) or deoxyxylulose phosphate (DXP), key intermediates of two independent pathways, the mevalonate and deoxyxylulose phosphate pathways. Plants exhibit the ability to utilize both pathways: the mevalonate pathway enzymes are compartmentalized in the cytosol and the deoxyxylulose in the chloroplast.21 Dimethylallyl pyrophosphate is then derived from isopentenyl pyrophosphate by means of allylic isomerization. This rearrangement is

12 catalyzed by the enzyme isopentenyl pyrophosphate isomerase.22 The mechanism is stereospecific and involves the removal of the HR proton at C-2 and addition of a proton from water at C -4 (Scheme 2.1).

HOOC OH

Mevalonic acid

Isopentenyl PP Isomerase OPP OPP

HR HS

Isopentenyl PP Dimethylallyl PP

O OH Deoxyxylulose P Scheme 2.1

The formation of geranyl pyrophosphate is catalyzed by the enzyme prenyl transferase and is accomplished by the head-to-tail addition of dimethylallyl pyrophosphate to isopentenyl pyrophosphate. The reaction most likely involves the ionization of DMAPP to the corresponding allylic cation. This transformation improves the electrophilicity of the substrate and aids in the alkylation of IPP. Electrophilic addition to the double bond of IPP yields a tertiary carbocation intermediate. Loss of the C-2 HR proton generates the monoterpenoid. The reaction is stereospecific and the E-geometrical isomer is formed exclusively (Scheme 2.2).21

13 -H+ OPP + OPP

HR HS

Dimethylallyl PP Allylic carbocation Isopentenyl PP

Prenyl transferase E

OPP 10 9

8 7 6 5 4 3 2 1 OPP

HR HS

Tertiary carbocation Geranyl PP intermediate Scheme 2.2

Most carbocyclic iridoids are derived from loganin (2.12) and have an 8-b-configuration. Conversion of geraniol to loganin is achieved in a stepwise manner and involves the following intermediates: 10-oxogeranial, iridodial, iridotrial and deoxyloganic acid.20

In the first step of the pathway geranial is converted to 10 -oxogeranial through a series of hydroxylations and oxidations. Cyclization of this dialdehyde yields iridodial. Further oxidation results in the formation of iridotrial that exists (in equlibrium) in two forms, a keto and hemiacetal form.20 Hemiacetal formation in iridotrial (keto form) leads to cyclization, so forming the heterocyclic ring of the iridoid skeleton. The next intermediate, deoxyloganic acid is formed by oxidation of the remaining aldehyde. Most iridoid s are glycosides and glycosylation transforms the hemiacetal into an acetal. Methylation of the carboxylic acid results in esterification and in the formation of deoxyloganin. Hydroxylation at C-7 of deoxyloganin yields loganin (Scheme 2.3).2

Decarboxylated iridoids such as aucubin (2.20) or antirrhinoside are formed in a similar manner to the carboxylated iridoids from 8-epi-loganin (2.24).20 The route includes the intermediates; 8-epi-iridodial, 8-epi-iridotrial and 8-epi-deoxyloganic acid.23,24 Decarboxylation of deoxyloganic acid yields the uncarboxylated iridoid intermediate. This is supported by the fact that in most decarboxylated iridoids the C-8 has an epi- configuration.13

14 hydroxylation CHO oxidations CHO Cyclization

CHO CHO

OH Geraniol 10-Oxogeranial Iridodial

oxidation

COOH CHO CHO H H hemiacetal formation CHO

O O CHO H H OGlc OH Deoxyloganic acid Iridotrial Iridotrial (hemiacetal) (keto)

CO 2CH3 H

HO Carboxylated iridoids O

H OGlc Loganin Scheme 2.3

2.2.3 Biological Activity of Iridoids

The use of plants in traditional medicines, from as early as ancient times has formed the foundation of what we call modern medicine today. Phytochemical investigations of plants used in traditional medicine have led to the discovery of many active compounds used in drugs. Iridoids are the main constituents of many Scrophulariaceae plant families

15 and subfamilies and they have a wide range of biological activities, but are most commonly known for their anti-inflammatory properties.

Devil’s claw, Harpagophytum procumbens (Pedaliaceae, subfamily of Scrophulariaceae) is indigenous to South Africa and is one of the most widely used traditional medicines.25 Harpagide (2.25) and harpagoside (2.26) were first isolated from H. procumbens in 1962.26 Procumbide (2.27) was isolated in 196427 and in 1983 three additional iridoid glycosides, procumboside (2.28), 8-O-p-coumarylharpagide (2.29) and 6’-O-p- coumaroylprocumbide (2.30) were isolated.28,29 Additional non-iridial compounds isolated for the first time from H. procumbens in ~ 1986 , include the phenolic glycosides acteoside (verbascoside) (2.8) and isoacteoside and the novel bioside b-(3’,4’,dihydroxy phenyl)ethyl-O-a-rhamnopyranosyl(1®3)-b-D-glucopyranoside.30

HO HO OH OH OH

O O O O O O H RO H H CH3 H3 C H3C O-Glc O-Glc O-Glc

2.25 R = H 2.27 2.28 2.26 R = cinnamoyl 2.29 R = p-coumaroyl

HO OH

OH O

H O O O HO OH HO O 2.30

The tuberized secondary roots of Devil’s claw are used in southern Africa to treat a variety of illnesses such as gastrointestinal problems, fever, birthing pains, rheumatic conditions, skin lesions and gout, etc.10 H. procumbens was introduced into Europe through Germany in the 1960’s, where it is mainly used an anti-inflammatory and anti- rheumatic herb to treat arthritis. The main active ingredients of the drug are the iridoids that constitute ~3 % of the total weight.25

16 There is conflicting data as to the effectiveness of the drug as an anti-inflammatory and analgesic agent. However, discrepancies in activity can be attributed to the fact that the compounds are deactivated under acidic conditions. It has, therefore, been suggested that oral administration is ineffective due to decomposition of the iridoids by gastric juices. Efficiency is significantly increased when the drug is given parenterally. Another contributing factor is the lack of standardization of the active constituent concentrations of herbal medicines.28,31

In some studies Harpagophytum has shown similar anti-inflammatory and analgesic properties to that of non-steroidal anti-inflammatory agents such as phenylbutazone and aspirin.32 A notable advantage is the minimal or even lack of potential side effects even when fairly high concentrations of Harpagophytum is administered.

Harpagophytum exhibits significant anti-oxidant activity and immunomodulatory activity that may also contribute to the anti-inflammatory activity.28 Another Harpagopytum species, namely H. zeyheri that also contains harpagoside (2.26) and 8-O-p-coumarylharpagide (2.29), exhibits similar anti-inflammatory activity. The two species are distinguished from each other by the ratio of harpagoside to 8-O-p-coumarylharpagide.33

The drug has also been used to treat a variety of digestive disorders. The iridoid glycosides cause reflex stimulation of the digestive processes and promote liver function leading to detoxification of the blood. A reduction in cholesterol levels has also been observed.31 Devil’s claw is contraindicated for pregnant woman because it has oxytoxic properties and has been used for regulation of the menstral cycle.10

Some iridoids also exhibit antiviral activity and this makes them potential antiviral drugs. Harpagoside (2.25) in addition to 8-O-acetylharpagide (2.31) and scorodioside (2.32) isolated from Schrophularia scorodonia have antiviral activity against the vesicular stomatitis virus (VSV). The iridoid scorodioside (2.32) exhibits moderate antiviral activity against the herpes simplex type-1 virus (HSV-1).34,35

Some iridoids are cytotoxic and thus have the potential to inhibit the growth of tumors and be used in the treatment of cancer. 8-O-Acetylharpagide (2.31) also isolated from Ajuga decumbens (Lamiaceae) exhibited strong anticancer activity against skin and hepatic tumors in mice.36

17 O

C OAc O

HO O H3 C HO O OH

O O

AcO H CH 3 OGlc HO OGlc

2.31 2.32

2.3 Literature Review on Flavonoids

2.3.1 Structural Classification

Flavonoids are major secondary plant metabolites and they all possess the same basic structural element, namely a 2-phenylchromane skeleton (2.33). Flavonoids are divided into two major groups according to the degree of saturation of the central heterocyclic ring. The unsaturated flavonoids have a planar geometry and include flavones, flavonols and anthocyanins. Saturated flavonoids are characterized by the absence of the 2,3- double bond and include flavanones and flavans. These molecules normally have one or more chiral centers. Most flavonoids occur naturally as water-soluble glycosylated derivatives and are frequently hydroxylated at the C-5, C-7 and 4’-positions.

8 O 1' 7 2

6 3 4 5 2.33

Flavones such as apigenin (2.34) and flavonols such as kaempferol (2.35) have the same basic structure, differing only by the presence of C-3 hydroxyl. Naringenin (2.36)

18 is a common flavanone and the biosynthe tic precursor to other flavonoid types. Dihydroflavonols such as (-)-taxifolin (2.37) are formed from flavanones (that have undergone hydroxylation). These structures all posses a carbonyl group at C-4 and are classified as 2-phenylchromones.

OH OH

HO O HO O

OH O OH O

2.34 2.35

OH OH

HO O HO O

OH O OH O

2.36 2.37

When there is no carbonyl group in the heterocyclic ring, flavonoids are classed as 2- phenylchromanes. Flavan-3-ols are commonly referred to as catechins e.g. (-)- epicatechin (2.38), an antioxid ant found in green tea.37 Flavan -3,4-diols have an additional 4-hydroxyl e.g. leucocyanidin (2.39). Oligomers of catechins may form, resulting in structures like the epicatechin trimer (2.40). These compounds are constituents of the tannins that con tribute to the astringency of foods and drinks.

19 OH OH

OH OH

HO O HO O

OH OH

OH OH OH 2.38 2.39

HO O

OH OH

OH HO O

OH OH

OH HO O

2.40

The remaining classes of flavonoids are anthocyanins (2 -phenylbenzopyrilliums), chalcones, aurones and isoflavanoids. Anthocyanins are plant pigments that frequently occur in conjunction with flavone or flavonol copigments and may contain metal cofactors.13 Delphinidin 3-gentiobiosyl, apigenin 7-glucosylmalonate (2.41) isolated from Eichhornia crassipes is an example of such a complex. In this complex the anthocyanin (part a), is covalently linked to the flavone (part b) by an O-b-D-glucose-(1®6)-glucose- malonylglucosyl bridge (R).38 This complex is responsible for the blue colour of the flower.

HO O OH OH

O R O O OH part a

OH O

part b 2.41

Chalcones such as naringenin chalcone (2.42) are the biosynthetic precursors to all flavonoids and contain an a,b-unsaturated ketone instead of the central heterocyclic ring.21 Hispidol (2.43) is an aurone and has a 2-benzylidenecoumaranone structure. In isoflavanoids such as genistein (2.44), the phenyl has migrated to the C-3 position on the heterocyclic ring forming a 3-phenylchromone.13

OH OH

HO OH HO O

O OH O

2.42 2.43

HO O

OH O OH

2.44

21 Modifications and structural diversification are achieved by variation in the hydroxylation pattern of the two aromatic rings, by methylation, glycosylation or prenylation. Some flavonoids such as liquiritigenin (2.45) may have lost a hydroxyl, formed by the reduction of naringenin-chalcone during biosynthesis.21 Glycosidic complexity can lead to considerable structural diversification because the type and combination of sugars attatched to different parts of the flavonoid can vary greatly. Additionally, one or more sugar moieties may be attached to different parts of the aglycone.13 C-glycosylated flavones in which the sugar and aglycone are directly linked by their carbons are commonly found in teas such as Aspalathus linearis (Rooibos tea). This tea is well known for its antispasmodic, antimutagenic and antioxidant properties and contains the C-glycosylated (S)- and (R)-diastereomers of eridodicytol-6-b-D-glucopyranoside, that differ only by the configuration of C-2 (2.46).39

OH OH

HO O HO O

Glc

O OH O 2.45 2.46

Flavonoids can also occur as dimers in which two flavonoids, mostly flavones and flavanones are joined together. These biflavonoids are products of phenol oxidative coupling and may be composed of the same or different types of flavonoids such as a flavone-flavone or flavone-flavanone complexes. They may be bonded together directly through their carbons most often by C -8 and C-6 or by a C-O-C interflavanyl link.13

2.3.2 Biosynthesis of Flavanones

Two main biosynthetic pathways contribute to the biosynthesis of flavonoids, the shikima te and the acetate pathways and they are responsible for the formation of chalcones, the basic precursors to all flavonoids.21 A chalcone (2.47) consists of a phenylpropanoid unit (p-coumaroyl-CoA or cinnamoyl-CoA) and a polyketide chain constructed from three units of malonyl-CoA that has been cyclized to a benzene.

22 Flavanones occur as intermediates as well as the end products of biosynthetic pathways.40 OH

B HO OH

O 4, 2',4',6'-Tetrahydroxychalcone

2.47

The shikimate pathway is the biosynthetic route to the aromatic amino acids L- phenylalanine, L-tyrosine and L-tryptophan. The pathway is initiated by the coupling of phosphoenolpyruvate with D-erythrose 4-phosphate in an aldol-type condensation. A series of transformations and additions results in the formation of the respective amino acids (Scheme 2.4).21 Cinnamic acid, the precusor of p-coumaroyl-CoA is formed from L-phenylalanine or L-tyrosine, the former being the preferred precursor.21 Deamination (E2 elimination) of the amino acid by the enzyme phenylalanine ammonia lyase yields cinnamic acid (Scheme 2.4).21

PEP CO2H CO H 2 CO2H P O

NH3 PO H E2 elimination

O HO

D-Erythrose-4-P L-Phenylalanine Cinnamic acid Scheme 2.4

Most flavanones are hydroxylated on the B-ring at the para -position. Therefore, the next step in the biosynthetic pathway is the transformation of the cinnamic acid to p-coumaric acid (4-hydroxycinnamic acid). The reaction is catalyzed by the enzyme cinnamate-4- hydroxylase together with NADPH and O2 as cofactors (Scheme 2.5). CoA ligase catalyzes the activation of p-coumaric acid to the corresponding thiol ester (p-coumaryl CoA) in a two-step reaction. In the first step the carboxylic acid is converted to an acyl-

23 AMP intermediate, through the phosphorolysis of ATP to AMP.41 The activated adenylate then binds with a low dissociation constant to the enzyme and is coupled to the thiol group of the CoA. Dissociation of the substrate from the enzyme gives the p- coumaryl-CoA and AMP (Scheme 2.5).42

CO2H CO 2H COSCoA

NADPH ATP/ Mg2+ AMP, PPi O2

Cinnimate-4- HSCoA hydroxylase

OH OH

Cinnamic acid p-Coumaric acid p-Coumaryl-CoA Scheme 2.5

Malonyl-CoA is formed by the carboxylation of acetyl-CoA. The reaction is catalyzed by acetyl-CoA carboxylase together with a biotin cofactor in the presence of ATP and Mg2+ (Scheme 2.6).22

O O O

- + H3C CoA + ATP + HCO3 HO CoA + ADP + Pi + H

Acetyl-CoA Malonyl-CoA Scheme 2.6

The formation of the chalcone is initiated by the sequential condensation of the starting unit of p-coumaroyl CoA (B-ring) with three malonyl-CoA units and is followed by the cyclization of the polyketide chain to form the second aromatic ring (Scheme 2.7). The reaction is catalyzed by chalcone synthase, a plant-specific polyketide synthase.43 The enzyme contains two active sites: the coumaryl binding pocket and the cyclization pocket that accommodates the polyketide chain. Nucleophilic attack of the phenol group (A- ring) on the a,b-unsaturated ketone results in formation of the central heterocyclic ring and thus the flavanone. The reaction is catalyzed by chalcone isomerase, is stereospecific and a single enantiomer is formed.44

24 COSCoA

O SCoA

3 Malonyl-CoA O

OH O O p-Coumaroyl-CoA Chalcone synthase

OH OH

HO O HO OH

Chalcone isomerase

OH O OH O (2S)-Naringenin Naringenin-chalcone (2.42) Scheme 2.7

2.3.3 Biological Properties of Flavonoids

Flavonoids are synonymous with the word colour. They are the pigments responsible for the colours of flowers, fruits and occasionally leaves. The yellow colour of flowers and fruits are derived from the chalcones and flavonols, whilst the anthocyanins give rise to the reds, blues and violets. Colourless flavonoids are also abundant and may function as copigments that either enhance the coloured flavonoids or absorb UV -radiation visible to insects thus aiding in pollination. These flavonoids also play an important role in the protection of the plant against the damaging effects of UV radiation.45, 46

The UV -absorbing characteristics of some flavonoids suggests that they can act as UV filters and thereby, offer protection against photooxidative damage. Flavonoids in the epiderm of the adaxial leaf surface, the epicuticular wax such as gnaphaliin (2.48) 47and even in leaf hairs e.g. kaempferol-3-O-b-D-(2”,6”-di-trans-p-coumaroylglucopyranoside (2.49 )48 protect the underlying photosynthetic tissue of plants.45 The general response of certain plants to an increase in exposure to UV radiation is an increase in flavonoid biosynthesis. This is supported by the fact that genetic mutants that lack epidermal

25 flavonoids, in plants such as Arabidopis do not respond well to UV radiation, whereas the wild-type responds well, even with changes in flavonoid type ratios.46,49 Flavonoids have a variety of other properties including anti-inflammatory, vascular, oestrogenic and cytotoxic antitumor activity.

OCH3

HO O HO O

O-Glc-(2",6"-di-trans-p-coumaroyl) OCH3

OH O OH O

2.48 2.49

One of the main functions of flavonoids as preformed compounds or through their accumulation as phytoalexins, is to protect plants from microbial invasion.50 Phytoalexins are compounds that are fo rmed in response to microbial or other invasions, almost the like the production of antibodies in an immune response. Naringenin (2.36) found in the heartwood of trees from the Rosaceae is an antifungal agent.51 The fungicidal properties of flavonoids are affected by phenolic substitution and in many cases it has been shown to decrease with increasing substitution.45 Isoflavonoids, flavanones and flavans are the most effective antimicrobial agents.

Flavonoids also have antiviral and antibacterial properties. The ever-increasing Aids epidemic has spurred the search for new and more effective antiviral drugs. Baicalin (5,6,7-trihydroxyflavone 7-glucuronide) (2.50) from Scutellaria baicalensis has a direct inhibitory effect on the virus.52 Other flavonoids such as the biflavonoid hinokiflavone inhibit enzymes required for viral replication, in this case HIV-1 reverse transcriptase.53 A derivative of quercetin (2.51), quercetin 3-(2”-O-galloylarabinopyranoside) (2.52) has anti-HIV properties as it inhibits the enzyme, HIV-1 integrase and interferes with the virus life cycle. Quercetin (2.51) itself also has an inhibitory effect on the virus.54,55

26 OH

R O HO O

HO R1

OH O OH O

2.50 R = O-glucuronide 2.51 R1 = OH 2.52 R1 = 2''-galloylarabinopyranoside

The ability of flavonoids to function as antioxidants has given them an important place in the field of human health and medicine. The radical scavenging ability of flavonoids is attributed to their structure and ability to interact with radical species [superoxide anion -· (O2 ), hydroxyl or peroxy radical] and sometimes singlet oxygen. The polyphenolic nature of flavonoids allows for the quenching of radical species. Furthermore, the metal chelating ability of flavonoids is another factor that contributes to antioxidant efficiency. Structural features important for metal chelation are the 4-carbonyl and 3 - or 5-hydroxyl and a catechol moiety in the B -ring. Quercetin flavonoids (2.51), common constituents of red wines, satisfy all of the above conditions and are therefore, good antioxid ants. Flavonoids also decrease membrane fluidity and this prevents leaking of harmful radicals to other cells.

Hesperidin is major byproduct of the citrus industry and functions as a mild anti- inflammatory with analgesic and antipyretic effects when given subcutaneously.45 Quercetin also exhibits anti-inflammatory activity.56 Phyto-oestrogen is a term given to non-steroidal plant compounds displaying oestrogenic properties. Oestrogenic flavonoids such as apigenin (2.34) and the isoflavonoid genistein (2.44) are able to bind to oestrogenic receptors. Isoflavonoids are abundant in leguminous plants and a diet high in soya and other legumes, may lead to the superfluous ingestion of oestrogenic flavonoids and this can lead to serious physiological problems.13,21 Many flavonoids such as pinocembrin 7-O-b-neohesperidoside (2.9) exhibit antitumor activity and this makes them potential anticancer drugs.57

The diverse array of biological functions of flavonoids opens the door to inumerous applications in the field of medicine for the treatment of disease and even general health maintenance. Diets rich in flavonoids may prove to be beneficial in the reduction of heart

27 disease, cancer etc, although it has also been shown that too much of a good thing can have the opposite effect.

2.7 References

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