Bioactivity Guided Isolation of Iridoid and Flavonoid Glycosides From Four Turkish Veronica Species

Bioactivity Guided Isolation of Iridoid

and Flavonoid Glycosides from

Four Turkish Veronica Species

Newaj Khan

4

th October 2010

This research project is submitted in part fulfillment of the requirements for the Master of

Pharmacy degree, University of London.

Department of Pharmaceutical and Biological Chemistry

Centre of Pharmacognosy

School of Pharmacy

University of London

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Acknowledgments

I would like to take this opportunity to first express my gratitude towards my supervisor

in Turkey. Dr. Şebnem Harput, who not only made my stay more pleasant, but was

always available when I needed any questions answered. I would also like to personally

thank all the pharmacognosy staff at Hacettepe University, without them I would not

have had the experience that I did.

I am grateful to my supervisor in the UK, Prof. Michael Heinrich, who has continuously

helped me develop my project even when times were hectic.

I thank the professors at Gazi University who helped to identify the plant specimens

used during this investigation and staff at Hacettepe who took the NMR readings.

Special thanks to Berni Widemann and Dr. Mire Zloh at the School of Pharmacy and

Prof. Gulberk Ucar at Hacettpe who organised my Erasmus placement and made the

opportunity a reality.

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Abstract

Background

Veronica has been used ethnomedicinally for the treatment of a number of ailments. The

use of Veronica for influenza, coughs, inflammation and rheumatic pains are to name

but a few of its reported uses. The species are said to contain a large number of iridoid

and flavonoid glycosides. It is thought, that these compounds are primarily responsible

for the treatment of the conditions mentioned.

The aims of this study were to investigate the antioxidant activity of the four Turkish

Veronica species V. chamaedrys, V. fuhsii, V. serpyllifolia and an unknown Veronica

species. We then carried out a bioactivity guided isolation to examine the chemical

composition of V. serpyllifolia further.

Method

2, 2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO) and superoxide (SO) radical

scavenging assays were carried out on the water extracts of the four species. We

calculated the percentage inhibition spectroscopically.

Column chromatography, medium performance liquid chromatography and vacuum

liquid chromatography were used to isolate compounds from V. serpyllifolia. DPPH

radical scavenging assays were used to help guide us.

Results

V. chamaedrys was found to be the most bioactive species, followed by V. serpyllifolia.

V. fuhsii was found to be the least active. Thin layer chromatography showed V.

chamaedrys to contain a large proportion of phenylethanoid glycosides. The remaining

species showed the presence of a large proportion of flavonoid glycosides.

Chromatography of V. serpyllifolia extract gave seven pure compounds VS1-VS7. 1H-

NMR spectroscopy was carried out on these compounds. VS2, VS3 and VS4 were

identified as verproside, catalposide and veronicoside, respectively. VS1 could not be

identified, possibly due to impurity. VS5-VS7 await spectral analysis.

Conclusion

V. serpyllifolia was found to contain the iridoid glycosides, verproside, catalposide and

veronicoside. The bioactivity assays suggest that Veronica species are effective

antioxidants.

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Contents

Introduction ..................................................................................................................... 6

The Genus Veronica ...................................................................................................... 7

Chemical Composition of Veronica .............................................................................. 9

Irirdoid Glycosides .................................................................................................... 9

Flavonoid Glycosides .............................................................................................. 10

Phenylethanoid Glycosides ...................................................................................... 13

Isolation and Purification of Compounds .................................................................... 16

Thin Layer Chromatography ................................................................................... 16

Column Chromatography ........................................................................................ 17

Medium Performance Liquid Chromatography ....................................................... 17

Vacuum Liquid Chromatography ............................................................................ 18

Bioactivity Tests .......................................................................................................... 18

DPPH Radical Scavenging Assay............................................................................ 19

NO Radical Scavenging Assay ................................................................................ 20

SO Radical Scavenging Assay ................................................................................. 20

Structure Elucidation ................................................................................................... 21

Aims ................................................................................................................................ 22

Method ........................................................................................................................... 23

Ethical and Safety Considerations ............................................................................... 23

Procedure Method ....................................................................................................... 23

Selecting a Species................................................................................................... 24

DPPH Radical Scavenging Assay ........................................................................ 25

SO Radical Scavenging Assay ............................................................................. 25

NO Radical Scavenging Assay ............................................................................ 26

Compound Isolation ................................................................................................. 26

Figure 12: Schematic of how and which fractions were obtained .............................. 30

Results ............................................................................................................................ 31

Selecting a Species ...................................................................................................... 31

TLC 1: Chromatographic comparison of the four Veronica species ....................... 31

Table 1: Radical scavenging assay of nitric oxide (NO) and superoxide (SO) by the

Veronica samples ..................................................................................................... 32

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Table 2: Radical scavenging assay of DPPH by the Veronica samples .................. 33

Compound Isolation .................................................................................................... 34

TLC 2: Chromatogram of eluted fractions from the polyamide column ................. 34

TLC 2.5: Chromatogram of combined polyamide fractions.................................... 35

Table 3: DPPH radical scavenging assay of the polyamide fractions ..................... 36

TLC 3: Chromatogram of eluted fractions from MPLC of Fr. 8-11........................ 37

TLC 3.6: Chromatogram of combined MPLC fractions from Fr. 8-11 ................... 39

TLC 4: Chromatogram of eluted fractions from VLC of Fr. 51-54 ........................ 39

TLC 5: Chromatogram of eluted fractions from MPLC of Fr. 22-28...................... 40

TLC 5.6: Chromatography of combined MPLC fractions from Fr. 22-28 .............. 42

TLC 6: Chromatogram of eluted fractions of Fr. 5-22 from a Sephadex column ... 42

TLC 7: Chromatogram of eluted fractions of Fr. 81-87 from a Sephadex column . 43

Structure Elucidation ................................................................................................... 43

TLC 8: Chromatogram of standard glucosides and isolated compounds ................ 43

Table 4: 1H-NMR spectroscopy findings for isolated compounds .......................... 44

Discussion ....................................................................................................................... 46

TLC and Scavenging Activity Assays ......................................................................... 46

Compound Isolation .................................................................................................... 47

1H-NMR structure elucidation ..................................................................................... 49

Free Radical Scavenging and its Importance in Inflammation ................................... 54

Conclusion ...................................................................................................................... 57

Further Work ................................................................................................................ 59

Appendix ........................................................................................................................ 60

1H-NMR Spectrum of VS1 .......................................................................................... 60




Bibliography .................................................................................................................. 77

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Course F

School of Pharmacy, University of London

PLAGIARISM STATEMENT

I, Newaj Khan, hereby confirm that the work submitted in this thesis is my own. Any ideas,

quotations, and paraphrasing from other peoples work and publications have been appropriately

referenced. I have not violated the School of Pharmacy’s policy on plagiarism.

Signature................................. Date..............................

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Introduction

Ethnomedicine is loosely defined as a discipline which investigates the traditional usage

of natural products, primarily herbal, by humans as medicines. Herbal remedies have

been used therapeutically for millennia. In fact, some records date their usage up to

60,000 years ago (Fabricant and Farnsworth, 2001).

A quarter of the drugs sold in developed, and three quarter in less developed countries

are thought to be based on naturally occurring compounds (Firn, 2003). Of all the plant-

derived compounds currently in worldwide use, the vast majority were identified via

leads on ethnomedicine (Sokmen et al, 1999).

The number of higher plant species on this planet is estimated at 250,000, and a mere

6% of these have been screened for biological activity (Fabricant and Farnsworth,

2001). Considering this inadequacy, the recent rise in the interest of ethnomedicine in

the developed world is not surprising (Firn, 2003).

When developing drugs from a plant origin, pharmaceutical companies isolate

chemicals for direct use. This is the case with medicines like digoxin, which is extracted

from the plant Digitalis lanata (Hollman, 1996). Sometimes compounds of known

structure can be used as leads to develop a new drug. Like the example of metformin

which was developed from guanidine, obtained from the plant Galega officinalis

(Witters, 2001). Alternatively, whole plants may be used for therapeutic benefit, like the

usage of Ginkgo biloba or the Veronica species (Fabricant and Farnsworth, 2001).

Turkey has one of the most diverse floras in continental Europe, it is home to more than

9000 flowering plant species. Being located between the east and the west, an extensive

knowledge of traditional medicine has accumulated there (Sokmen et al, 1999). Of these

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9000 flowering species 79, are represented by the genus Veronica, of which 26 are

endemic to Turkey (Harput et al, 2002c).

The Genus Veronica

The genus Veronica, commonly known as speedwell, previously belonged to the

Scrophulariaceae family, however, due to the recent analysis of the DNA sequence it

has been re-classified to the family Plantaginaceae (Munoz-Centeno et al, 2006). The

species are mainly distributed in Europe and Asia, particularly in the Mediterranean

region. Others species are found in Africa and North America (Lahloub et al, 1993).

Traditionally, species of Veronica have been used for a number of therapeutic remedies.

In China V. anagallis-aquatica has been used for the treatment of influenza,

haemoptysis, laryngopharyngitis and hernia (Su, Zhu and Jia, 1999). Fujita et al, 1995

described its use for treating abdominal and rheumatic pains in Anatolia. V. persica is

used for the treatment of cancer in Peru (Graham et al, 2000). The use of V. hederifolia

for coughs and influenza and V. polita for the use as an expectorant in Turkey has also

been reported (Tomassini, 1995). Other species are said to be effective remedies for

wound healing and as a diuretic (Baytop, 1984). It is worthwhile noting that the vast

majority of these conditions are closely related to inflammation.

The species of Veronica to be studied during this investigation are V. serpyllifolia, V.

chamaedrys, V. fuhsii and an unknown Veronica species. These belong to the subgenera

Beccabunga, Chamaedrys and Pentasepalae respectively, and are illustrated in a

phylogenetic tree on figure 1 (Albach et al, 2004). Species with a closer phylogenetic

relationship are known to contain similar chemical compositions.

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Veronica species are said be made up of primarily iridoid glycosides, although, the

presence of a number of flavonoid and phenylethanoid glycosides have also been

reported. (Harput et al, 2002c).

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Chemical Composition of Veronica

Irirdoid Glycosides

The bitter taste attributed to certain plants is often due to the presence of iridoids

(Rodriguez et al, 1998). Iridoids are found as natural constituents in a large number of

plant families, usually in the form of a glycoside. In 1958 Halpern and Schmid, upon

investigating the compound plumieride, showed that iridoids are characterized by their

cyclopentan [c] pyran monoterpenoid substructure, as illustrated on Figure 2 (El-Naggar

and Beal, 1980). Cleavage of the cyclopentane ring produces a group of compounds

known as the seco-iridoids and cleavage of the pyran ring produces iridoid derivatives.

Iridoids are said to be the structural link between terpenes and alkaloids (Dinda et al,

2007).

O

OGly

Figure 2: Iridoid Glycloside Skeleton

Cyclopentane Ring

Pyran Ring

Recent studies have shown that iridoids exhibit a range of biological activities, such as

antinflammatory, immunomodulatory, neuroprotective, hepatoprotective,

cardioprotective, anticancer, antimicrobic and many others (Tundis et al, 2008). We will

be focusing on the antioxidant activity in particular during this investigation.

Over the years a number of iridoid compounds have been isolated from a variety of

plants, including those of the Veronica species. Their structures were identified via

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TLC, NMR, UV and IR spectroscopy. In fact, there have been a number of iridoid

review articles written that name all the iridoids identified to date (Dinda et al, 2007).

Jensen et al, 2005 examined a number of Veronica species to study their iridoid

glycoside composition. They found that the composition of the genus is relatively

homogeneous. They also identified that a standard Veronica species contains aucubin,

catalpol, 6-O-catalpol esters and possibly one or more carboxylated iridoids (Figure 3).

However, there was some discrepancy when investigating V. pocilla and V.

chamaedrys. Where, V. pocilla was shown to contain some unusual compounds whereas

V. chamaedrys showed very little iridoid content.

OO

OOH

Glc

OH

O

OOH

Glc

OH

OO

OOH

Glc

O

O

R

Aucubin Catalpol 6-O-Catalpol Ester

Figure 3: Iridoid Glycosides found in Veronica Species

Flavonoid Glycosides

Flavonoids are polyphenolic compounds found widely in fruits and vegetables and are

said to be responsible for the exuberant colours visible on the flowers and fruits of

plants (Brouillard and Cheminat, 1988).

The basic flavonoid structure contains the flavan nucleus. This consists of 15 carbon

atoms arranged in three rings, as illustrated on figure 4. Flavonoids are classed

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according to the substitution and oxidation level within the middle ring, whereas,

compounds from within a class are distinguished due to the substitution within the

remaining two rings (Pietta, 2000).

8

5

7

6

2

3

O

4

Figure 4: Flavonoid Skeleton

There are a number flavonoid classes of which flavones, flavanones, isoflavones,

flavonols, flavanonols and flavan-3-ols are of particular interest (Pietta, 2000).

Isoflavones have an aromatic ring attached to carbon 3 in the middle ring as opposed to

that on the diagram at carbon 2. Flavones and flavonols are enantiomers of flavanones

and flavanonols respectively. Figure 5 illustrates these classes.

O

O

O

O

OH

O

O

OH

O

O

O

O

O

OH

Flavone Flavanone Flavonol

Flavanonol Isoflavone Flavan-3-ol

Figure 5: Different Flavonoid Classes

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Middleton, 1998 reported the useful biological activities of flavonoids. In vitro

experiments showed that flavonoids exhibited free radical scavenging, anti-

inflammatory, antiallergic, antiviral and anticarcinogenic properties. Ibrahim et al, 2007

also reported the analgesic and diuretic activity of flavonoids. The activities appear to

be similar to those of the iridoids.

The flavonoid chemistry of Veronica has been extensively studied (Taskova et al,

2008). They appear in plants primarily in their glycosylated form (Jadhav et al, 2008).

In the Veronica species, they usually showed glycosylation at the 5th

or 7th

carbon

(Saracoglu et al, 2004). On studying the Veronica species in New Zealand, Taskova et

al, 2008 revealed that the majority of flavonoids present in Veronica are flavone

glycosides.

The main compounds extracted from various Veronica species by Grayer in 1978 were

also flavone glycosides, where the flavone groups were luteolin and apigenin. They also

demonstrated the presence of methylated flavones like chrysoeriol, tricin, acacetin and

6-hydroxy derivatives of these compounds. The glycoside group was found to be either

glucose or glucuronic acid in most cases. These chemicals have been illustrated on

figure 6.

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O

OOH

OH

OH

OH

Figure 6: Flavonoid Glycosides found in Veronica Species

O

OOH

OH

OH

O

OOH

OH

OH

O

O

OOH

OH

O

OH

O O

OOH

OH

O

Luteolin Apigenin Chrysoeriol

Tricin Acacetin

Hydroxylation is sometimes found

at position 6

Glycosylation occurs at postion

5 or 7

Phenylethanoid Glycosides

Phenylethanoid glycosides are a group of polyphenolic compounds spread throughout

the plant kingdom, most of which are isolated from medicinal plants (Jimenez and

Riguera, 1994). They are found predominantly in the Verbenaceae, Lamiaceae and

Scrophulariaceae families (Aydın et al, 2004).

Structurally, phenylethanoid compounds are distinguished by a glucopyranose molecule

bound to a hydroxyphenylethyl moiety via a glycosidic linkage. Often, the structure also

contains aromatic acid moieties such as caffeic, ferulic or cinnamic acid attached via an

ester linkage. Numerous other sugars like rhamnose and xylose may also be bound to

the core glucose residue via a glycosidic bond (Fu, Pang and Wong, 2008). This has

been illustrated with the phenylethanoid glycoside plantamajoside in figure 7.

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OH

OH

OH

OH

O

O

OH

OH

OH

OH

O

OH

OH

O

O

O

Figure 7: Plantamajoside a Phenylethanoid Glycoside

Dihydroxyphenylethyl moiety

Glucopyranose Core Residue

Glycosidic Bond

Ester Bond

Caffeoyl Moiety

Glucopyranose Sugar Group

Glycosidic Bond

There can be an attachment of increasing numbers of different sugars that may be bound

to different carbons on the glucose residue. Therefore, these chemicals can be extremely

complex and difficult to categorise.

Fu, Pang and Wong, 2008 have also recorded a wide range of biological activities for

phenylethanoid glycosides. A number of both in vivo and in vitro investigations have

found them to exhibit antioxidant, free radical scavenging, neuroprotective,

hepatoprotective, cardioprotective, antimicrobial, anti-inflammatory,

immunomodulatory and analgesic activities.

Several studies have been carried out previously on the Veronica species to identify

their phenylethanoid glycoside content. In 2007, Kostadinova et al isolated

phenylethanoid triglycosides, turrilliosides A and B, from V. turrilliana. Another

investigation successfully extracted persicoside, acteoside, isoacteoside and

lavandulifolioside from V. persica (Harput et al, 2002b). Isopersicoside and two

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unnamed compounds were also found in this species by Aoshima et al, 1994b. Research

on the species V. fuhsii showed the presence of plantamajoside and fuhsioside (Ozipek

et al, 1999).

Ehrenoside and verpectoside A, B and C were extracted from V. pectinata var.

glandulosa (Saracoglu et al, 2002). Another unnamed compound was extracted from V.

undulata but was later named isochionoside B by Taskova et al, 2010 (Aoshima et al,

1994a). Fourteen new phenylethanoid glycosides were extracted from V. thomsonii and

V. pulvinaris by Taskova et al, in 2010.

Review articles on the general phenylethanoid composition of the Veronica species are

rather limited; this is possibly due to the largely varying phenylethanoid content within

the genus, as demonstrated with the examples aforementioned.

During this investigation we will be studying in particular the iridoid and flavonoid

glycoside composition of our chosen Veronica species. Veronica is said to contain

mainly iridoid glycosides (Saracoglu et al, 2002). Therefore, any reported

ethnomedicinal properties of the species would most probably be due to the presence of

these compounds. Hence, we have chosen to study this component further.

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Isolation and Purification of Compounds

To be able to elucidate the chemical composition of a species it is required to first

isolate pure compounds from a plant specimen. Extraction of compounds is usually

carried out using solvents such as ethanol and methanol as they are both cheap and

readily available (Suomi et al, 2000). Typically, this is carried out at room temperature

however there have been some reports to the use of hot ethanol (Iossifova et al, 1999).

During our investigation we will be using methanol at room temperature for extraction.

Chromatographic methods are used to isolate pure compounds. This usually involves

separation by a mobile phase on a stationary phase. Meier and Sticher, 1977 have

mentioned that the most common methods of separation are thin layer chromatography

(TLC), column chromatography (CC), gas chromatography (GC), and high-performance

liquid chromatography (HPLC). More recently, the use of medium-performance liquid

chromatography (MPLC) and vacuum liquid chromatography (VLC) have also been

reported (Ersoz et al, 2007). Of these, we will only be using TLC, CC, MPLC and VLC.

GC is not often used for iridoid or flavonoid glycoside separation and the lack of HPLC

equipment means we will not be using these two techniques.

Thin Layer Chromatography

Before any extraction can take place we must be able to detect the presence of our

compounds of interest, in this case iridoid and flavonoid glycosides. TLC is the method

used. This involves the separation of compounds using a solvent system on a plate

containing a stationary phase. This is either made of silica gel or Sephadex. The plates

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are then exposed using a reagent, which is chosen depending on the compound being

separated.

The reagent used by Lahloub et al, 1993 is the one which is most commonly used for

iridoid glycosides; this is the one which we will be using. Here, vanillin-H2SO4 is

sprayed on to chromatography plates for exposure of iridoid compounds. Alternatively,

FeCl3 dissolved in ethanol can be used for exposure (Si et al, 2008).

Column Chromatography

Column chromatography is the traditional and most employed method for compound

isolation, especially for the initial stages of chromatography. Often polyamide, alumina,

silica gel and Sephadex columns are used as the stationary medium (Marston and

Hostettmann, 1991). For the extraction of iridoids and flavonoids; polyamide, silica gel

and Sephadex are the more preferred methods. Polyamide requires extraction via

gradient elution with increasing methanol concentration in water. In Sephadex

chromatography only methanol is used to wash out the sample (Ersoz et al, 2002).

Usually, chloroform or dichloromethane and methanol are eluted in a gradient when

using a silica gel column (Garg et al, 1994).

Medium Performance Liquid Chromatography

MPLC is a suitable method for the rapid pressurised separation of relatively polar

compounds such as iridoid and flavonoid glycosides. The stationary medium used for

this is usually reverse phase (RP) silica gel, and a mixture of methanol and water is the

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employed method for elution (Marston and Hostettmann, 1991). There have been a

number of reports of successful separations using this technique. Roby and Stermitz,

1984 isolated the iridoid glycoside penstemonoside from Castilleja rhexifolia. Lahloub

et al, 1993 also separated a number of iridoid glycosides from V. anagallis-aquatica

using this method.

Vacuum Liquid Chromatoghraphy

VLC involves the rapid elution of compounds under a vacuum. It is also a suitable

method for the isolation of iridoids and flvaonoids. This procedure often showed

excellent efficiency, with very little sample lost in process (Pelletier, Chokshi and

Desai, 1986). The usage of silica gel or RP-silica gel as the stationary medium seems to

be predominant. Handjieva et al, 1991 isolated iridoids from Valeriana officinalis using

this medium. However, there have also been reports for use of polyamide and Sephadex

using this technique (Akdemir et al, 2004) (Akbay et al, 2003).

Bioactivity Tests

To be able to decide on which species of Veronica to investigate and also to guide us on

which compounds to extract, it is necessary for us to examine which extracts are the

most active. As mentioned before, Veronica is said to have a number of activities,

however we will be studying the antioxidant component further.

The antioxidant activity of a specimen can be examined in a number of ways. Most

often the scavenging activity of the sample is tested against some free radicals. The use

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of DPPH, superoxide (SO), nitric oxide (NO), hydroxyl and peroxide radicals have been

reported (Schinella et al, 2002) (Saracoglu et al, 2002) (Tsai et al, 2007). During our

investigation we will be using DPPH, SO and NO radical scavenging assays.

DPPH Radical Scavenging Assay

DPPH or 2, 2-diphenyl-1-picrylhydrazyl is a stable radical used to test the antioxidant

activity of an extract. The radical is absorbed at the wavelength 515nm (Brand-

Williams, Cuvelier and Berset, 1995). Therefore, if the absorbance is recorded before

and after the addition of extract to a solution of DPPH, depending on the level of

decolouration, the radical scavenging activity can be revealed. The reaction taking place

is illustrated on figure 8.

NO2

NO2O

2N

N*

N

NO2

NO2O

2N

N

N

HAntioxidant-H +

DPPH- Purple

Antioxidant* +

DPPH-H Colourless

Figure 8: Reaction of DPPH radical with Antioxidant Compound

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NO Radical Scavenging Assay

NO radicals are formed from sodium nitroprusside. However, to detect the scavenging

activity directly is not possible, as the absorbance cannot be detected without the

presence of a dye. Often, Griess’ reagent is added to the solution for this. The nitric

oxide radicals cause the diazotization of the sulphanilamide present in the reagent; this

is followed by coupling with naphthyl ethylenediamine, also present within the reagent,

causing a purple colouration (Mulla et al, 2009). As before, the absorbance at 577nm

before and after can be used to detect the level of antioxidant activity (Harput et al,

2002a).

NH2

SO3H

N2+

SO3H

NH

NH2

+ NHO3S NH

NH2

N

NO

Diazonium Salt

Azo Dye (Purple)

Sulphanilamide Naphthyl ethylenediamine

Therefore, a reduction in NO radicals leads to reduced absorbance

Figure 9: Mechanism of Action of Griess' Reagent

SO Radical Scavenging Assay

Superoxide radicals (O2-) were successfully produced when air-saturated dimethyl

sulphoxide (DMSO) was mixed with NaOH (Hyland et al, 1983). Nitro blue tetrazolium

(NBT) indicates a blue colour when reduced by the superoxide radical. This allows us to

detect how much of the superoxide radicals have been scavenged by the extract.

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Therefore, examining the absorbance at 560nm will allow us to determine the

bioactivity of the compound (Logan, Hammond and Stormo, 2008).

Structure Elucidation

The most common methods used for structure elucidation are through UV, IR, mass,

1H-NMR and

13C-NMR spectroscopic methods. Previously, there has also been the

usage of chemical methods for identification (Aldjia, 2004). We will be studying the

1H-NMR spectra of our compounds to elucidate their structures.

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Aims

- To determine which Veronica species to investigate further from analysing the

TLC results and bioactivity assays.

- To isolate pure iridoid and flavonoid glycosides from the chosen plant species

with the aid of bioactivity assays.

- To carry out NMR spectroscopy and analyse the findings to identify these

compounds.

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Method

Ethical and Safety Considerations

Standard safety guidelines for laboratory work were followed. Appropriate laboratory

coats and safety goggles were worn at all times. Desk areas were kept clean and dry

throughout the experiment. Bottles were closed when not being used to avoid spillage.

Any spillages that did occur were immediately wiped up and any glassware broken was

immediately discarded of to avoid slipping and injury. Hands were washed thoroughly

after leaving the laboratory to avoid carrying contaminants. Bottles and solvent systems

were labelled with the name, date and contents to avoid confusion and handling them

appropriately.

Volatile toxic compounds such as petroleum ether and chloroform we handled in well

ventilated areas. A fume cupboard was used when spraying TLC plates with corrosive

aerosolized vanillin-H2SO4. Exposure to skin was avoided by using spatulas when

transferring irritants such as nitro blue tetrazolium (NBT) and naphthyl ethylenediamine

dihydrochloride. Dermal exposure to methanol, chloroform and petroleum ether was

also avoided by using pipettes and measuring cylinders.

Procedure Method

The method of this project can be split into two parts. Part one is where we decided

which Veronica species to study further, and part two is the isolation of pure

compounds from the chosen species.

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Selecting a Species

Four different Veronica species were taken from the Abant region of Bolu in Turkey

during May 2009. Of these, species 2 has not been identified. Species 1 is V.

chamaedrys L (herbarium number: HUEF 09326), species 3 is V. fuhsii (herbarium

number: HUEF 09327), and species 4 is V. serpyllifolia (herbarium number: HUEF

09328). V. fuhsii has previously been studied by professors at Hacettepe University.

A methanol extraction was carried out on the air dried aerial parts of each of the four

specimens. This was then followed by thin layer chromatography (TLC) to help identify

which species to study further. TLC was carried out in the solvent system CHCl3:

CH3OH: H2O in the ratio 70:30:3 in a Camag glass cabin (22 x 23 x 8 cm). This solvent

system was used for all the TLC plates throughout the investigation. Pre-coated,

commercial silica gel plates (Merck, 60F254) were used for TLC.

The TLC plates were examined under UV light (at 254nm and 366nm) using a Camag

UV lamp and were further exposed using vanillin-H2SO4. The samples were then

evaporated under a vacuum and the crude extract was obtained. The crude extract was

then re-dissolved in distilled water and partitioned with petroleum ether to remove the

non-polar chlorophyll. The aqueous phase was freeze-dried using a Virtis Freezemobile

5 and a dry sample was obtained.

We also decided to carry out numerous bioactivity assays by investigating the

antioxidant activity of the different species. With this we were able to determine which

sample had the greatest activity, also aiding us in deciding which sample to study

further.

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DPPH Radical Scavenging Assay

A proportion of the four Veronica samples were dissolved in methanol to produce

solutions of 200, 100, 50 and 25μg/ml concentration. Enough solution was made for

four repeat experiments.

1mM DPPH solution was produced from its powder where 7.886mg was dissolved in

methanol to produce 20ml of solution.

200μl of each sample at each concentration were pipetted into individual wells on a well

plate. Each concentration was repeated four times for more accurate findings. 200μl of

methanol was also added into the remaining wells to act as the blank. 50μl of DPPH

solution was then added to each well. The plate was then incubated for 30minutes

followed by a recording of the absorbance at 520nm.

SO Radical Scavenging Assay

A proportion of the four Veronica samples were dissolved in dimethyl sulfoxide

(DMSO) to produce solutions of 800, 400, 200, 100 and 25μg/ml concentration.

NBT was dissolved in DMSO to form a 1mg/ml concentration solution. Alkaline

DMSO was produced by mixing in a ratio of 9 parts DMSO to 1 part 5mM NaOH

solution.

30μl of each sample at each concentration were pipetted into individual wells on a well

plate, this was repeated four times for each concentration. 30μl of DMSO was used to

act as the blank. 100μl of alkaline DMSO was then added to each well followed by the

addition of 10μl NBT solution. The absorbance was then recorded at 560nm.

P a g e | 26

NO Radical Scavenging Assay

Veronica solutions of concentrations 800, 400, 200, 100 and 25μg/ml were obtained by

dissolving samples in methanol.

Griess reagent was produced by dissolving 1g sulfanilamide and 0.1g naphthyl

ethylenediamine dihydrochloride in 2.5ml aqueous dihydrogen phosphate. The solution

was then made up to 100ml with distilled water.

60μl of each sample at each concentration were pipetted four times into individual wells

on a well plate. 60μl of methanol was used to act as the blank. 60μl of 10mM sodium

nitroprusside dissolved in phosphate buffer saline was added to each well. The plates

were then incubated for 150minutes under light. 120μl of Griess reagent was then added

and the absorbance was then recorded at 577nm.

The equation % Inhibition= ((A0-Aabs)/A0) x100, where A0 is the absorbance of the

blank and Aabs is the absorbance with the compound, was used to calculate the

percentage inhibition.

Compound Isolation

We decided to investigate V. serpyllifolia further. 93g of dried plant was milled,

extracted, partitioned and freeze-dried as before. The extract was then re-dissolved in

water with the aid of a sonicator (Transsonic 570) and fractionated through a polyamide

column (50-160 μm). The sample was eluted out of the column with an increasing

methanol concentration (from 0- 100%) and fractions were collected. TLC was then

carried out on all these fractions, which were then combined according to their chemical

P a g e | 27

constituents by examining the plates under UV. These fractions were then evaporated

under vacuum and re-dissolved in water. The fractions were then lyophilised and the

mass was recorded.

Figure 10: The polyamide column

Picture drawn on OpenOffice.org 3.2 Draw

Polyamide Column

Cotton wool to prevent polyamide being eluted

Sample injected on top of column

Test tubes collecting elution

Fractions

Methanol added in increasing concentration

Cotton wool preventing agitation of polyamide on adding methanol

A TLC was carried out on the combined fractions to see which fraction to study further.

We also carried out the DPPH radical scavenging activity test on these fractions to see

which the most active were, so guiding us on which fractions to isolate from. However,

this time we only experimented with the concentration 200μg/ml of sample.

Medium pressure liquid chromatography (MPLC) was then carried out on the chosen

fractions (Fr. 8-11 and Fr. 22-28). Büchi (25 mmx460 mm) glass column filled

Lichroprep RP-18 were used during MPLC.

P a g e | 28

This works very similarly to the polyamide column. However, the stationary medium in

this case is different and the solution is eluted under pressure using a pump (Büchi B-

684). The methanol is mixed with water and added mechanically, rather than manually.

As before, TLC was carried out on the eluted fractions and those showing similar

constituents were combined. This was followed by the same steps aforementioned;

evaporation, dissolving and lyophilisation, followed by a TLC on the fractions. When

pure compound had been isolated, which can be determined by looking at the TLC

plates, approximately 8mg of the sample was sent off for 1H-NMR spectroscopy. The

pure samples were labelled VS1-VS7. Spectroscopy was carried out in the solvent

DMSO at the frequency 400MHz using the JEOL JNM-A 500.

Figure 11: Sample TLC plate

Picture drawn on OpenOffice.org 3.2 Draw

Blotted Samples

Similar constituents so can be combined

Pure compound.

Sent for NMR

Impure compound. Requires further

fractionation

We decided to do a TLC on the isolated pure compounds with some common standard

iridoid glucosides. Using this we were able to figure out, along with the 1H-NMR

findings, what the isolated compounds were.

P a g e | 29

This method is continuously repeated until the most abundant compounds have been

isolated. Other column chromatography techniques may also be used. A Sephadex

column may be used to isolate compounds with greatly differing molecular weights. A

column may be put under vacuum to help separate compounds. Both of these methods

have been used during the course of this investigation and will be discussed further in

the discussion section. To isolate all the compounds in a plant is a lengthy process; we

managed to extract only seven during this experiment.

All the chemicals used during this investigation were received from Sigma-Aldrich Co

(St. Louis, MO) other than sulfanilamide and naphthyl ethylenediamine

dihydrochloride, which were received from Merck Co. (Darmstadt, Germany).

All chemical structures in this thesis were drawn on ACDLabs 12.0 ChemDraw. Images

were drawn on OpenOffice.org 3.2 Draw and graphs and schematic were drawn on

Microsoft Office 2007 Excel.

P a g e | 30

Figure 12: Schematic of how and which fractions were obtained

Drawn on Microsoft Office Excel 2007

Dried Aerial Parts of V. serpyllifolia

93g

MeOH Extract19.55g

MeOH Extraction

Petroleum Ether Extract Water Extract

Petroleum Ether/ Water

Polyamide CCMeOH:H20 (0-10-25-50-75-100)

Fr. 34.5555g

Fr. 41.9694g

Fr. 5-74.3746g

Fr. 8-111.1396g

Fr. 12-150.4419g

Fr. 16-210.4194g

Fr. 22-280.7575g

Fr. 29-340.4991g

Fr. 35-420.2082g

Fr. 43-480.0457g

Fr. 36171.6mg

Fr. 41-4616.8mg

Fr. 479.8mg

Fr. 49-5015.8mg(VS3)

Fr. 51-5473.5mg

Fr. 123.6mg

Fr. 44.7mg

Fr. 1-2892mg

Fr. 55-6928.6mg

Fr. 70-749mg

(VS4)

Reverse Phase- MPLCMeOH:H20 (15-50)

Fr. 6-125mg

Fr. 350.5mg

Fr. 33-340.7mg

Fr. 24-262mg

Fr. 13-2262.5mg

Reverse Phase- VLCMeOH:H20 (25-50)

Fr. 33-40558.6mg

(VS2)

Fr. 29-3238.9mg(VS1)

Fr. 8-111.1396g

Fr. 5-2215.3mg

Fr. 27-3014.1mg

Fr. 37-4050.6mg

Fr. 42-44104.5mg

Fr. 49-59131.1mg

Fr. 73-7747.1mg

Fr. 81-8772.1mg

Fr. 98-10021.1mg(VS5)

Fr. 106-11034.1mg(VS6)

Reverse Phase- MPLCMeOH:H20 (15-70)

Fr. 2-64.4mg

Fr. 7-117mg

Fr. 5-73.5mg

Fr. 8-1231.8mg

Fr. 13-1420.7mg

(VS7)

Fr. 15-172.6mg

Sephadex- CC

MeOH 100%

Sephadex- CC

MeOH 100%

Fr. 22-280.7575g

P a g e | 31

Results

Selecting a Species

TLC 1: Chromatographic comparison of the four Veronica species

TLC of the methanol extract of each species was carried out. V. chamaedrys showed an

orange glow under UV inspection. V. serpyllifolia showed a strong yellow glow. V.

fuhsii and the unknown Veronica showed a slight yellow glow.

P a g e | 32

Table 1: Radical scavenging assay of nitric oxide (NO) and superoxide (SO) by the

Veronica samples

The mean % inhibition and the standard deviation have been calculated for both tests to

see which samples are the most active.

Concentration of Sample/ μg/ml

800 400 200 100 25

Mean

%

Inh.

SD

Mean

%

Inh.

SD

Mean

%

Inh.

SD

Mean

%

Inh.

SD

Mean

%

Inh.

SD

V.

chamaedrys

NO 58 1.225 37 2.243 15 3.347 1.9 8.696 -1.2 6.271

SO 76 0.8798 72 1.074 66 1.842 53 2.880 35 6.731

Unknown

Veronica

NO 52 0.7605 33 5.950 18 4.811 11 11.26 5.2 14.17

SO 46 3.405 9.2 4.834 3.7 4.128 4.4 3.264 3.5 5.050

V. fuhsii NO 30 3.446 17 3.201 4.8 1.947 -0.24 5.031 -3.4 2.817

SO 60 0.849 23 3.011 15 4.057 -13 2.343 3.2 8.257

V.

serpyllifolia

NO 53 0.7213 33 5.879 27 3.003 20 0.6682 0.62 2.372

SO 42 2.208 41 4.810 23 3.893 4.9 3.872 -17 5.341

P a g e | 33

Table 2: Radical scavenging assay of DPPH by the Veronica samples

The mean % inhibition and the standard deviation have been calculated for the DPPH

scavenging activity test to see which samples are the most active.

Concentration of Sample/ μg/ml

200 100 50 25

Mean %

Inh SD

Mean %

Inh SD

Mean %

Inh SD

Mean %

Inh SD

V. chamaedrys 90 1.984 86 4.028 45 5.085 21 1.823

Unknown

Veronica 91 0.2020 73 1.048 43 2.009 25 4.905

V. fuhsii 79 0.5941 47 5.685 26 6.300 12 0.5682

V. serpyllifolia 91 0.09470 88 1.021 49 1.410 24 3.377

P a g e | 34

Compound Isolation

TLC 2: Chromatogram of eluted fractions from the polyamide column

Fractionation was conducted on V. serpyllifolia using a polyamide column and TLC

carried out.

P a g e | 35

TLC 2.5: Chromatogram of combined polyamide fractions

Fractions with similar content were combined and TLC carried out.

P a g e | 36

Table 3: DPPH radical scavenging assay of the polyamide fractions

DPPH scavenging activity test was carried out on the fractions to see which were most

active.

Mean % Inh SD

Fr. 4 22 2.650

Fr. 5-7 32 0.8320

Fr. 8-11 58 2.269

Fr. 12-15 61 2.675

Fr. 16-21 60 2.070

Fr. 22-28 64 1.118

P a g e | 37

TLC 3: Chromatogram of eluted fractions from MPLC of Fr. 8-11

It was decided to study Fr. 8-11 and Fr. 22-28 further. Fractionation was carried on Fr.

8-11 first using MPLC. Below are the TLC plates for these fractions.

P a g e | 38

P a g e | 39

TLC 3.6: Chromatogram of combined MPLC fractions from Fr. 8-11

Fractions were combined, as before, pure samples VS1- VS4 were obtained.

TLC 4: Chromatogram of eluted fractions from VLC of Fr. 51-54

VLC was carried out on Fr. 51-54 of Fr.8-11 to isolate compounds. TLC results were

recorded.

P a g e | 40

TLC 5: Chromatogram of eluted fractions from MPLC of Fr. 22-28

TLC results of the MPLC fractions of Fr. 22-28 of the polyamide fraction.

P a g e | 41

P a g e | 42

TLC 5.6: Chromatography of combined MPLC fractions from Fr. 22-28

Fractions were combined, as before, pure samples VS5 and VS6 were obtained.

TLC 6: Chromatogram of eluted fractions of Fr. 5-22 from a Sephadex column

Fractionation was conducted on Fr. 5-22 of Fr. 22-28 using a Sephadex column.

P a g e | 43

TLC 7: Chromatogram of eluted fractions of Fr. 81-87 from a Sephadex column

Fractionation was conducted on Fr. 81-87 of Fr. 22-28 using a Sephadex column. VS7

was obtained.

Structure Elucidation

TLC 8: Chromatogram of standard glucosides and isolated compounds

P a g e | 44

Table 4: 1H-NMR spectroscopy findings for isolated compounds

NMR analysis has yet to be carried out on the isolated flavonoid glycosides (VS5, VS6,

and VS7). Below are the findings for VS1- VS4.

Please see appendix for full 1H-NMR spectra of VS1-VS4.

(DMSO; 1H: 400 MHz)

C

Number

VS2 VS3 VS4

δH/

ppm

J/ Hz δH/

ppm

J/ Hz δH/

ppm

J/ Hz

Aglycone

1 CH 5.11 d 9.4 CH 5.11 d 9.5 CH 5.13 d 9.6

3 CH 6.43 dd 5.9, 1.6 CH 6.43 dd 5.9, 1.7 CH 6.44 dd 5.9, 1.8

4 CH 4.95 dd 5.9, 4.3 CH 4.96 dd 5.8, 4.4 CH 4.99 dd 5.8, 4.5

5 CH 2.54 m CH 2.55 m CH 2.60 m

6 CH 5.05 dd 7.9, 0.9 CH 5.06 dd 8.1, 1.0 CH 5.14 dd 8.0, 1.8

7 CH 3.6-3.8

m

CH 3.6-3.8

m

CH 3.6-3.8

m

8 C C C

9 CH 2.47 m CH 2.46 m CH 2.33 m

10a CH2 3.92 d 13.2 CH2 3.92 d 13.3 CH2 3.93 d 13.3

10b CH2 3.6-3.8

m

CH2 3.6-3.8

m

CH2 3.6-3.8

m

Sugar

1’ CH 4.62 d 7.9 CH 4.62 d 7.8 CH 4.63 d 7.8

2’

CH 3.0-3.3

m

CH 3.0-3.3

m

CH 3.0-3.3

m

3’

4’

5’

6’a CH2 3.6-3.8

m

CH2 3.6-3.8

m

CH2 3.6-3.8

m

6’b CH2 CH2 CH2

Aromatic

1” C C C

2” CH 7.41 d 2.0 CH 7.85 d 6.9 CH 8.02 d 7.1

3” C CH 6.85 d 8.7 CH 7.57 t 7.9

4” C C CH 7.70 t 7.5

5” CH 6.83 d 8.3 CH 6.85 d 8.7 CH 7.57 t 7.9

6” CH 7.37 d 9.4 CH 7.85 d 6.9 CH 8.02 d 7.1

VS1- 1H-NMR (400 MHz, DMSO) δ: 7.34 m, 7.14 d J= 8.3 Hz, 6.78 m, 6.43 m, 6.26 m,

5.10 d J= 9.456 Hz, 5.02 d J= 7.73 Hz, 4.95 m, 4.62 d J= 7.9, 3.92 d 13.1, 3.6-3.8 m,

3.0-3.3 m, 2.67 m, 2.33 m.

P a g e | 45

4'

2'3'

1'

5' 6'O

O

65

98

7

1 O

3

4

O

10a,b O

O

OHOH

OH

OH

OH

OH

OH

1"

2"3" 4"

5"

6"a,b

Figure 13: VS2 - Verproside

4'

2'3'

1'

5' 6'O

O

65

98

7

1 O

3

4

O

10a,b O

O

OHOH

OH

OH

OH

OH

1"

2"3" 4"

5"

6"a,b

Figure 14: VS3 - Catalposide

4'

2'3'

1'

5' 6'O

O

65

98

7

1 O

3

4

O

10a,b O

O

OHOH

OH

OH

OH 1"

2"3" 4"

5"

6"a,b

Figue 15: VS4 - Veronicoside

P a g e | 46

Discussion

TLC and Scavenging Activity Assays

TLC of the four species revealed an orange glow for V. chamaedrys, this is indicative of

phenylethanoid glycosides. This finding is supported by Jensen et al, 2005, where V.

chamaedrys was found to contain very little iridoid content and high levels of

phenylethanoids. The yellow glow given off of the remaining three species is due to the

presence of flavonoid glycosides. V. serpyllifolia showed a more prominent yellow

glow than the other two species (TLC1). As the aim of this project was to isolate

flavonoid and iridoid glycosides, the latter was favoured.

The radical scavenging activity assays all generally showed the same thing. We find that

V. chamaedrys is the most effective at scavenging both nitric oxide and superoxide free

radicals. At 400 μg/ml of sample we see that V. chamaedrys inhibited the NO free

radicals by 37%, which was then followed by V. serpyllifolia and the unknown

Veronica species at 33%. V. fuhsii was found to inhibit a mere 17%.

The trend is similar with the SO assay at the same concentration. The DPPH scavenging

activity test showed V. serpyllifolia to be the most active but closely followed by V.

chamaedrys.

There was an anomaly present in the SO results. At 100 μg/ml of V. fuhsii we see a

plunge in the scavenging activity, with a lower bioactivity than that at 25 μg/ml of

extract (Graph 2). We carried the experiment in quadruplicates but all the repeats

seemed to be effected. The most probable explanation for this would be that the 100

μg/ml sample was not transferred to the wells.

P a g e | 47

Our results showed us that V. chamaedrys is the most active species and is followed by

V. serpyllifolia. V. serpyllifolia, however, showed the presence of a large amount of

iridoid and flavonoid glycosides, as confirmed by Taskova et al, 2002. Therefore, we

decided to isolate from this species.

Previously, there have been very few investigations carried out in comparing the

bioactivity of the different Veronica species. Only one investigation has been reported;

were V. polita was found to have the most radical scavenging activity (Harput et al,

2002b). The report found the species to be almost as effective as BHA as an antioxidant.

Compound Isolation

The species extract was fractionated through a polyamide column using methanol. This

is the most suited method and is often used to isolate flavonoid and iridoid glycosides.

Its mode of action is due to the adsorbance of compounds through hydrogen bonding

(Strack et al, 1975).

The TLC plates of the fractions obtained from the polyamide fractions showed that

fraction 22-28 contained a high number of flavonoid glycosides (TLC 2.5). This

fraction also exhibited the greatest radical scavenging activity with 64% inhibition of

DPPH radicals. Fraction 8-11 contained a large number of iridoid glycosides, and was

relatively clean. Therefore, these fractions were chosen to be isolated from.

These two fractions underwent MPLC through Lichroprep RP-18. The surface of the

stationary medium contains non-polar alkyl chains. Therefore, as inferred by the name

less polar compounds are eluted first, opposite to the polyamide column.

P a g e | 48

VS1 to VS4 were obtained from fraction 8-11; Rf values 0.61, 0.56, 0.67 and 0.74

respectively. We also decided to put fraction 51-54 of fraction 8-11 through VLC in

small RP-18 silica gel column to separate the three compounds present on the TLC plate

(TLC 3.6). Eluting through a normal column may not be enough to separate the

compounds and MPLC on such a small amount of compound may not elute a large

enough amount of compound for NMR spectroscopy. VLC was carried out, but we were

unsuccessful in out attempts of isolation as shown on TLC 4.

TLC 5.6 shows us the fractions obtained from fraction 22-28. VS 5 (Rf 0.75) and VS6

(Rf 0.75) were isolated, although the samples were somewhat unclean, reliable NMR

results could still be obtained. We saw that fraction 5-22 was impure and required

further isolation to remove these impurities. The impurities are difficult to see on the

plate as exposure was not very successful. However, they could be seen distinctly under

UV light at Rf 0.2. We chose to carry out fractionation in a Sephadex column to produce

a purer sample. A very small amount of compound was extracted from the column

(4.4mg and 7mg). Therefore, NMR could not be carried out in the JEOL JNM-A 500,

which we were using. However, these compounds may be examined in the future by

more advanced equipment.

TLC 7 shows the fractions obtained from Sephadex chromatography of fraction 81-87

of fraction 22-28. We can see on TLC 5.6 that there are two compounds very close

together here (Rf 0.6, and 0.65), we attempted to separate them. From the Sephadex

column eluate we extracted VS7 (Rf 0.5), although it is difficult to understand from the

TLC whether we isolated a pure compound or not.

TLC of the extracted compounds against some standard compounds show us matching

Rf values of VS2, VS3 and VS4 with verproside, catalposide and veronicoside

P a g e | 49

respectively (TLC 8). The marking for VS1 could not be distinguished properly,

although it seems to be lower than before relative to the marking for VS2. VS5-VS7 are

all flavonoids and cannot be compared to the standards.

Upon carrying out chromatography on our TLC plates we noticed that the flavonoid

glycosides were sometimes very difficult to detect, as can be seen on TLC 5 and TLC 8.

We were using vanillin-H2SO4 for exposure of the plates, which is more suited for

iridoid glycosides. The use of reagents such as NH3, AlCl3, Al2(SO4)3, ZrOCl2,

diphenylboric acid or EDTA may have been of more benefit (Heirmann and Bucar,

1994).

Throughout the experiment we had a fluctuating set of standard deviations for the

radical scavenging assays, indicating to us that our results obtained were not very

reliable. Further statistical analysis has not been carried out because this was not

essential for our experiment. The assays were intended to just guide us towards which

fractions to study further therefore, an extremely reliable set of results was not

necessary.

1H-NMR structure elucidation

Past studies have shown that the Veronica species are rich in iridoid glucosides, mainly

aucubin, catalpol, benzoic and cinnamic acid esters of catalpol (Harput et al, 2003).

Comparison of our spectra with the aforementioned will enable us to determine which

our compounds may be related to.

On comparison we see that VS1-VS4 have a number of correlating peaks. We see peaks

at around δ 5.11, 6.43, 4.95, 2.54, 5.05, 3.92, and 4.62 with correlating J values. This

P a g e | 50

indicates to us that they have similar chemical structures. The peaks on the far left,

deshielded region must be in an aromatic or heterocyclic system as the chemical shifts

are between δ 6 and 9 (Field et al, 2008).

All three compounds had a doublet peak at approximately δ 5.1, this is characteristic of

the acetylic proton H1 found in iridoids. They also contain a double doublet around δ

6.4, with J values of approximately 6.0 and 1.5 Hz. This indicates to us that there is no

substitution at C4 and C5 (Aldjia, 2004).

The spectra can be compared to that of the peaks for aucubin and catalpol to get a better

understanding of the structure. Although the experiment by Roby and Stermitz, 1984

was carried out in the solvent D2O at 320 MHz we see that there is a good match of

peaks and J values with that of catalpol. H1-H9 of the aglycone moiety of catalopol has

a chemical shift of δ 5.02 d (J= 9.8), 6.33 dd (J= 6.0, 1.7), 5.08 dd (J= 6.0, 4.6), 2.25 m,

4.00 dd (J= 8.1, 1.0), 3.56, and 2.58 dd (9.8, 7.7). Our compound was dissimilar to

aucubin as the H7 chemical shift was too high, at δ 5.78, for aucubin; ours was at δ 3.6-

3.8. This is due the lack of the electron withdrawing oxygen atom bound to C7 and C8

on the cyclopentane ring.

Figure 16: Chemical Structure of Catalpol and Aucubin

P a g e | 51

Therefore, we have established that our compounds all have the catalpol skeleton in

common within their structures. We now know that our compounds are most probably

benzoic esters of catalpol due to the aromatic signals present on our spectra. The

compound cannot be a cinnamic ester of catalpol as the peak for the olefinic protons are

not present on the spectra. We would expect an extra peak more deshielded than H2” to

represent the olefinic H7”, this can be seen by studying the spectra of cinnamic acid

(Liu, Li and Sun, 2004).

The aromatic portion of the compounds was then studied separately. For VS4 we saw

five protons in this region with only three peaks (2H, 2H and 1H). Due to the symmetry

we could match the protons with the correct proton number. δ 8.02 with C2” and C6”, δ

7.57 with C3” and C5”, and δ 7.70 with C4”.The J-values were all approximately 7,

therefore, confirming that these protons were ortho to each other. This was further

confirmed by comparison with the resonances of benzoic acid (Pretsch, Buhlmann and

Badertscher, 2009).

Figure 17: Peaks for Benzoic Esters

VS3 had four protons but also showed symmetry as there were only two peaks δ 7.85

and 6.85, therefore, we could assume that C4” had a group attached to it. This was

P a g e | 52

confirmed by the similarity with that of the spectrum 4-methyl-paraben (δ 7.7 and 6.65)

(Hazarika, Parajuli and Phukan, 2007).

Figure 18: Peaks for 4-Hydroxy Benzoic Esters

VS2 showed three peaks for three protons (δ 7.41, 6.83 and 7.37). The J values for these

peaks were 2.0, 8.3 and 9.4 respectively. This means that the δ 7.41 peak must be meta

to another proton and the other two protons must be ortho to each other. Using this

knowledge we could place the protons onto C2”, C5” and C6”. The spectrum

corresponded with that of protocatechuic acid (δ 7.52, 6.89 and 7.46) which confirmed

this hypothesis (Wang and Gao, 2009).

Figure 19: Peaks for 3, 4-Hydroxy Benzoic Esters

P a g e | 53

From analysing just the proton NMR spectrum of VS1 we were not able to determine

what the structure of the compound is. It is possible that this compound is impure

therefore, the spectra is very unclear. TLC of VS1 is also ambiguous. Further

experimental analysis needs to be carried out to elucidate the structure of this

compound.

Taskova et al in 2002 extracted a number of iridoid glycosides from V. serpyllifolia.

Catalpol, aucubin, verproside, veronicoside, catalposide, amphicoside and verminoside

were identified by NMR spectroscopy. VS1 could possibly be one of these compounds

or a mix of two.

The NMR findings matched that of TLC 8. We see that VS2 was matched with

verproside, VS3 was matched with catalposide and VS4 was matched with

veronicoside. These three compounds contain within their structures the skeletons of

protocatechuic acid, paraben and benzoic acid respectively, which are bound to catalpol

via an ester bond.

The structure can be further confirmed by comparing the NMR findings to that of the

actual compounds found in literature. The three compounds VS2, VS3 and VS4

matched that which was recorded by Kwak et al, 2009 and Sticher and Afifi-Yazar,

1979. Research carried out by Taskova et al, 2002 also shows the successful extraction

and identification of these three compounds from the species V. serpyllifolia.

Extensive research has been carried out on a number of Veronica species, including

bioactivity assays. Although the iridoid content of the species V. serpyllifolia has

previously been examined the bioactivity and flavonoid content of the species had yet to

be studied. Although, we were not able to carry out spectral analysis on the flavonoid

P a g e | 54

glycosides extracted, we managed to successfully isolate them. As the flavonoid content

for this species has not been studied before, there is potential for the discovery of new

compounds.

Free Radical Scavenging and its Importance in Inflammation

During the course of this experiment we carried out radical scavenging activity assays

on the Veronica extracts. The importance of radical scavenging activity is said to be

linked to that of the ethnomedicinal use of the plant for wound healing (Baytop, 1984).

It is well known that nitric oxide (NO) is an important mediator in the inflammatory

process; often large amounts of NO radicals produced by inducible NOS are pro-

inflammatory (Guzik et al, 2003). Therefore, scavenging of these radicals should

theoretically prevent inflammation.

We found that the Veronica samples were effective NO radical scavengers, indicating to

us that they would prevent inflammation. This is the first time such assays have been

comparatively discussed on whole species from the genus Veronica. However, a

number of experiments have been carried out on components extracted from certain

species.

Kwak et al, 2009 successfully isolated a number of iridoid glycoside from V. peregrina.

Antioxidant activity was tested prior to the isolation of the compounds on the three

partitions (chloroform, ethanoic acid and n-butanol) of the methanol extract of the plant.

The ethanoic acid fraction showed greater antioxidant activity than the control, trolox an

antioxidative vitamin E analogue (Forrest et al, 1994).

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Saracoglu et al, 2002 carried out a DPPH radical scavenging activity test on four

phenylethanoid glycosides isolated from V. pectinata. Two of these compounds were

found to have a more potent activity than BHA and the other two were shown to have a

more modest activity, but an activity nevertheless. Other phenylethanoid compounds

extracted from V. turrilliana by Kostadinova et al in 2007 were also found to have

strong radical scavenging activity; of greater potency than quercetrin.

These experiments showed us that chemicals within these species had a profound

antioxidant activity. However, the investigations that were carried out were only

conducted in-vitro. Although the anti-inflammatory action of the species had been

discussed, their action had not been established other than through ethnomedicinal uses

and indirect and theoretical evidences.

The first step towards establishing the anti-inflammatory action of Veronica was carried

out by Recio et al, 1994. We know that the genus Veronica contain a large amount of

iridoid glycosides. So, if a link between iridoid and anti-inflammatory action could be

established one could assume that the species would have some anti-inflammatory

activity.

In 1994 Recio et al carried out experiments using carrageenan to induce a paw oedema

and TPA to induce an ear oedema in mice. Following external application, a number of

iridoids, including catalpol derivatives and aucubin, like those extracted during our

experiment, were found to have potent anti-inflammatory activity. Jia, Hong and

Minter, 1999 also carried out an investigation on catalpol analogues and found a similar

response.

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In 2002b Harput et al then set out to prove that the reported anti-inflammatory activity

was due to the scavenging activity of Veronica and not due to the lack of transcription

of iNOS. As noted before, iNOS is primarily responsible for producing NO which is

thought to be responsible for aggravating inflammation (Guzik et al, 2003). This would

prove to us that the radical scavenging activity of Veronica is the cause of the anti-

inflammatory action.

A series of experiments were carried out on mouse macrophages. Macrophages are

responsible for producing NO through enzymes iNOS, eNOS and nNOS; different

isoforms of the nitric oxide synthase enzyme. They concluded that the anti-

inflammatory action was due to the radical scavenging activity of NO, as the expression

of iNOS was not altered. Positive DPPH radical scavenging activity confirmed this

hypothesis. This finding was supported by our research; we found that NO free radicals

were scavenged by our Veronica samples.

Eventually, in 2005 Kupeli et al showed a directly proved the anti-inflammatory action

of Veronica. In-vivo activity of iridoids had been demonstrated before, however, this

was the first time in-vivo experimentation had been carried out with just the plant

specimen.

Inflammation was induced by injecting carrageenan into a mouse paw. Veronica

anagallis-aquatica extract was then applied topically to the paw to see what the effect

was over a given time against the control. A statistically significant reduction in

inflammation was noticed when compared to that of the control. This study proves that

the assumption that Veronica has anti-inflammatory properties that have been used

ethnomedicinally for wound healing.

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Conclusion

After carrying out three radical scavenging activity tests we found V. chamaedrys on

average to be the most potent out of the four species V. serpyllifolia, V. chamaedrys, V.

fuhsii and unknown Veronica. Chamaedrys was found to be the most active in both

nitric oxide and superoxide radical scavenging tests, indicating to us a greater anti-

inflammatory activity. TLC analysis showed the presence of a large number of

phenylethanoid glycosides, a notion supported by the work carried out by Jensen et al in

2005.

We found V. serpyllifolia on average to be the second most active species, the DPPH

scavenging activity test showed it to be the most potent. TLC analysis showed the

presence of a large number of flavonoid and iridoid glycosides as supported by Taskova

et al, 2002. V. fuhsii was found to be the least active of the four species.

V. serpyllioflia was studied further and fractionation was carried out on the sample.

Following fractionation through a polyamide column, MPLC, VLC and Sephadex

column pure compounds VS1-VS7 were obtained.

1H-NMR spectroscopy of the four compounds showed the peaks for containing catalpol

in their sub-skeleton. Further analysis of the aromatic region of VS2 showed a match

with protocatechuic acid (Wang and Gao, 2009), VS3 with paraben (Hazarika, Parajuli

and Phukan, 2007) and VS4 with benzoic acid (Pretsch, Buhlmann and Badertscher,

2009).

Therefore, we identified VS2 as verproside, VS3 as catalposide and VS4 as

veronicoside. This was further established from the match obtained from TLC of our

compounds against the known compounds. Our 1H-NMR findings correlated with those

P a g e | 58

reported by Kwak et al, 2009 and Sticher and Afifi-Yazar, 1979. Research carried out

by Taskova et al, 2002 shows the successful extraction and identification of these three

compounds from the species V. serpyllifolia.

Structure elucidation however of VS1 was unsuccessful. The spectrum obtained was

relatively unclear. It is possible that VS1 is impure therefore a definite structure could

not be revealed. The flavonoid compounds VS5-VS7 await spectral analysis and

structure elucidation.

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Further Work

As the spectral data obtained for VS2-VS4 were very clean and a compound could be

identified no further work needs to be carried out on these samples. Since VS1 could

not be identified and the spectrum was unclear 1H-NMR spectroscopy may be carried

out again. Further TLC on a larger plate may identify whether the sample is pure or

impure.

1H-NMR and

13C NMR spectroscopy may be carried out on VS5-VS7 and if necessary

IR spectroscopy to obtain the molecular mass of the compound. Having more

spectroscopic methods of identification of the compounds will make structure

elucidation easier.

As only a small number of compounds have been isolated, as can be seen from the TLC

plates, work may be carried out to isolate and identify further pure compounds from the

remaining fractions of the species. This includes a number of flavonoid, iridoid and

phenylethanoid glycosides.

Comparative in-vivo assays may be carried out with the four Veronica species on an

inflammation induced mouse paw. This would enable us to see which plant extract is

the most effective in-vivo for anti-inflammatory activity.

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Appendix

1H-NMR Spectrum of VS1

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Bioactivity Guided Isolation of Iridoid and Flavonoid Glycosides From Four Turkish Veronica Species

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