Bioactivity Guided Isolation of Iridoid

and Flavonoid Glycosides from

Four Turkish Veronica Species

Newaj Khan
4th 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. ebnem 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. 1HNMR 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
1

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

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

Conclusion...................................................................................................................... 57
Further Work ................................................................................................................ 59
Appendix ........................................................................................................................ 60
1

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

H-NMR Spectrum of VS2.......................................................................................... 65

H-NMR Spectrum of VS3.......................................................................................... 69

H-NMR Spectrum of VS4.......................................................................................... 73

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 Pharmacys 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 plantderived 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).

Figure 2: Iridoid Glycloside Skeleton

Cyclopentane
Ring

Pyran Ring

O
O

Gly

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.

Figure 3: Iridoid Glycosides found in Veronica Species

O
HO

O
HO

HO

O
O

O
HO

Glc
Aucubin

O
Glc
Catalpol

HO

O
Glc
6-O-Catalpol Ester

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).

Figure 4: Flavonoid Skeleton

8
7

O
2

3
5

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.

Figure 5: Different Flavonoid Classes

OH
Flavone

Flavanone

Flavonol

O
OH

Flavanonol

Isoflavone

OH
Flavan-3-ol

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

experiments showed that flavonoids exhibited free radical scavenging, antiinflammatory, 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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Figure 6: Flavonoid Glycosides found in Veronica Species

OH
OH

HO

HO

OH

HO

OH

Luteolin

OH

HO

OH

Apigenin

Chrysoeriol

O
OH

HO

Glycosylation
occurs at postion
5 or 7

HO

Tricin

HO

Hydroxylation is
sometimes found
at position 6

OH

Acacetin

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 (Aydn 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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Figure 7: Plantamajoside a Phenylethanoid Glycoside

Caffeoyl Moiety

Glucopyranose
Core Residue

Ester Bond

Glycosidic Bond

O
OH
O

HO
O
HO
HO
HO

OH
O

OH

OH

OH
Glycosidic Bond

OH

Dihydroxyphenylethyl moiety

Glucopyranose
Sugar Group

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

P a g e | 15

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

P a g e | 17

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

P a g e | 18

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

P a g e | 19

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 (BrandWilliams, 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.

Figure 8: Reaction of DPPH radical with Antioxidant Compound

N*

Antioxidant-H +

Antioxidant* +

NO 2

O 2N

NO 2
DPPH- Purple

N H
NO 2

O 2N

NO 2
DPPH-H Colourless

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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).

Figure 9: Mechanism of Action of Griess' Reagent

NH2

N2

NH2

NO
HN

+
SO 3H
Sulphanilamide

HO3S

NH
NH2

SO 3H
Diazonium
Salt

Naphthyl
ethylenediamine

Azo Dye
(Purple)

Therefore, a reduction in NO radicals leads to reduced absorbance

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.

P a g e | 21

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,
1

H-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
1

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

P a g e | 22

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.

P a g e | 23

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 25g/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.
200l 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. 200l of
methanol was also added into the remaining wells to act as the blank. 50l 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 25g/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.
30l of each sample at each concentration were pipetted into individual wells on a well
plate, this was repeated four times for each concentration. 30l of DMSO was used to
act as the blank. 100l of alkaline DMSO was then added to each well followed by the
addition of 10l 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 25g/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.
60l of each sample at each concentration were pipetted four times into individual wells
on a well plate. 60l of methanol was used to act as the blank. 60l of 10mM sodium
nitroprusside dissolved in phosphate buffer saline was added to each well. The plates
were then incubated for 150minutes under light. 120l 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 m). 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
Methanol added in
increasing
concentration

Cotton wool preventing

agitation of polyamide
on adding methanol

Sample injected on
top of column

Polyamide Column

Cotton wool to
prevent
polyamide
being eluted

Test tubes
collecting elution

Fractions

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 200g/ml of sample.
Medium pressure liquid chromatography (MPLC) was then carried out on the chosen
fractions (Fr. 8-11 and Fr. 22-28). Bchi (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 (Bchi B684). 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

Similar constituents
so can be combined

Impure
compound.
Pure
compound. Requires
further
Sent for
fractionation
NMR

Blotted Samples

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
Extraction

MeOH Extract
19.55g
Petroleum Ether/
Water

Petroleum Ether Extract

Water Extract
Polyamide CC
MeOH:H20 (0-10-25-50-75-100)

Fr. 3
4.5555g

Fr. 4
1.9694g

Fr. 5-7
4.3746g

Fr. 8-11
1.1396g

Fr. 12-15
0.4419g

Fr. 16-21
0.4194g

Fr. 22-28
0.7575g

Fr. 29-34
0.4991g

Fr. 35-42
0.2082g

Fr. 43-48
0.0457g

Fr. 8-11
1.1396g
Reverse Phase- MPLC
MeOH:H20 (15-50)

Fr. 1-28
92mg

Fr. 4
4.7mg

Fr. 12
3.6mg

Fr. 29-32
38.9mg
(VS1)

Fr. 33-40
558.6mg
(VS2)

Fr. 36
171.6mg

Fr. 41-46
16.8mg

Fr. 47
9.8mg

Fr. 49-50
15.8mg
(VS3)

Fr. 51-54
73.5mg

Fr. 55-69
28.6mg

Fr. 70-74
9mg
(VS4)

Reverse Phase- VLC

MeOH:H20 (25-50)

Fr. 6-12
5mg

Fr. 13-22
62.5mg

Fr. 24-26
2mg

Fr. 33-34
0.7mg

Fr. 35
0.5mg

Fr. 22-28
0.7575g
Reverse Phase- MPLC
MeOH:H20 (15-70)

Fr. 5-22
15.3mg

Fr. 27-30
14.1mg

Fr. 37-40
50.6mg

Fr. 42-44
104.5mg

Fr. 49-59
131.1mg

Fr. 73-77
47.1mg

Fr. 81-87
72.1mg

Sephadex- CC
MeOH 100%

Fr. 2-6
4.4mg

Fr. 7-11
7mg

Fr. 98-100
21.1mg
(VS5)

Sephadex- CC
MeOH 100%

Fr. 5-7
3.5mg

Fr. 8-12
31.8mg

Fr. 13-14
20.7mg
(VS7)

Fr. 15-17
2.6mg

Fr. 106-110
34.1mg
(VS6)

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.

800

V.
chamaedrys
Unknown
Veronica
V. fuhsii
V.
serpyllifolia

NO
SO
NO
SO
NO
SO
NO
SO

Mean
%
Inh.
58
76
52
46
30
60
53
42

Concentration of Sample/ g/ml

200
100
Mean
Mean
SD
%
SD
%
SD
Inh.
Inh.
2.243
15
3.347
1.9
8.696
1.074
66
1.842
53
2.880
5.950
18
4.811
11
11.26
4.834
3.7
4.128
4.4
3.264
3.201
4.8
1.947
-0.24
5.031
3.011
15
4.057
-13
2.343
5.879
27
3.003
20
0.6682
4.810
23
3.893
4.9
3.872

400
SD

1.225
0.8798
0.7605
3.405
3.446
0.849
0.7213
2.208

Mean
%
Inh.
37
72
33
9.2
17
23
33
41

25
Mean
%
Inh.
-1.2
35
5.2
3.5
-3.4
3.2
0.62
-17

SD
6.271
6.731
14.17
5.050
2.817
8.257
2.372
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.

200

V. chamaedrys
Unknown
Veronica
V. fuhsii
V. serpyllifolia

Concentration of Sample/ g/ml

100
50
Mean %
Mean %
SD
SD
Inh
Inh
86
4.028
45
5.085

Mean %
Inh
90

1.984

91

0.2020

73

1.048

43

79
91

0.5941
0.09470

47
88

5.685
1.021

26
49

SD

25
Mean %
Inh
21

1.823

2.009

25

4.905

6.300
1.410

12
24

0.5682
3.377

SD

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.

Fr. 4
Fr. 5-7
Fr. 8-11
Fr. 12-15
Fr. 16-21
Fr. 22-28

Mean % Inh

SD

22
32
58
61
60
64

2.650
0.8320
2.269
2.675
2.070
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)
VS2
H/
ppm

C
Number
Aglycone
1
3
4
5
6
7

CH
CH
CH
CH
CH
CH

8
9
10a
10b

C
CH
CH2
CH2

Sugar
1
2
3
4
5
6a
6b
Aromatic
1
2
3
4
5
6

C
CH
C
C
CH
CH

5.11 d
6.43 dd
4.95 dd
2.54 m
5.05 dd
3.6-3.8
m
2.47 m
3.92 d
3.6-3.8
m

CH

4.62 d

CH
CH2
CH2

VS3
H/
ppm

J/ Hz

9.4
5.9, 1.6
5.9, 4.3
7.9, 0.9

13.2

7.9

CH
CH
CH
CH
CH
CH
C
CH
CH2
CH2

5.11 d
6.43 dd
4.96 dd
2.55 m
5.06 dd
3.6-3.8
m
2.46 m
3.92 d
3.6-3.8
m

CH

4.62 d

3.0-3.3
m

CH

3.6-3.8
m

CH2
CH2

7.41 d

2.0

6.83 d
7.37 d

8.3
9.4

C
CH
CH
C
CH
CH

VS4
H/
ppm

J/ Hz

9.5
5.9, 1.7
5.8, 4.4
8.1, 1.0

13.3

7.8

CH
CH
CH
CH
CH
CH
C
CH
CH2
CH2

5.13 d
6.44 dd
4.99 dd
2.60 m
5.14 dd
3.6-3.8
m
2.33 m
3.93 d
3.6-3.8
m

CH

4.63 d

3.0-3.3
m

CH

3.0-3.3
m

3.6-3.8
m

CH2
CH2

3.6-3.8
m

C
CH
CH
CH
CH
CH

8.02 d
7.57 t
7.70 t
7.57 t
8.02 d

7.85 d
6.85 d

6.9
8.7

6.85 d
7.85 d

8.7
6.9

J/ Hz

9.6
5.9, 1.8
5.8, 4.5
8.0, 1.8

13.3

7.8

7.1
7.9
7.5
7.9
7.1

VS1- 1H-NMR (400 MHz, DMSO) : 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

Figure 13: VS2 - Verproside

HO
HO

3'

2'

4'

1'
5'

6'

O
HO

O
OH
6"a,b

10a,b

1"

5"

OH
3"

2"

4"

OH

HO

Figure 14: VS3 - Catalposide

3'

HO

2'

4'

1'
5'

6'

O
HO

OH
6"a,b

10a,b

1"

5"

OH
3"

2"

4"

OH

HO

Figue 15: VS4 - Veronicoside

3'
4'

2'

1'
5'

6'

6
5

O
HO

8
10a,b

O
OH

6"a,b
1"

5"

O
2"

HO

OH
3"

OH

4"

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 g/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 g/ml of V. fuhsii we see a
plunge in the scavenging activity, with a lower bioactivity than that at 25 g/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
g/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.

H-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 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 6 and 9 (Field et al, 2008).
All three compounds had a doublet peak at approximately 5.1, this is characteristic of
the acetylic proton H1 found in iridoids. They also contain a double doublet around
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 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 5.78, for aucubin; ours was at 3.63.8. 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. 8.02 with C2 and C6,
7.57 with C3 and C5, and 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 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 ( 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 ( 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 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 ( 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 proinflammatory (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).

P a g e | 55

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.

P a g e | 56

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 antiinflammatory 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 antiinflammatory 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.

P a g e | 57

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 antiinflammatory 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.
1

H-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.

P a g e | 59

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.
1

H-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.

P a g e | 60

Appendix
1

H-NMR Spectrum of VS1

P a g e | 61

P a g e | 62

P a g e | 63

P a g e | 64

P a g e | 65

H-NMR Spectrum of VS2

P a g e | 66

P a g e | 67

P a g e | 68

P a g e | 69

H-NMR Spectrum of VS3

P a g e | 70

P a g e | 71

P a g e | 72

P a g e | 73

H-NMR Spectrum of VS4

P a g e | 74

P a g e | 75

P a g e | 76

P a g e | 77

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