Medicinal Activity of viscosa —A preliminary study —

RIRDC Publication No. 08/172

ESSENTIAL OILS AND EXTRACTS

RIRDCInnovation for rural Australia

Medicinal Activity of — a preliminary study

by Andrew Pengelly

November 2008

RIRDC Publication No 08/172 RIRDC Project No PRJ-000703

© 2008 Rural Industries Research and Development Corporation. All rights reserved.

ISBN 1 74151 761 3 ISSN 1440-6845

Medicinal Activity of Dodonaea viscosa - a preliminary study

Publication No. 08/172 Project No. PRJ-000703

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Researcher Contact Details Andrew Pengelly The University of Newcastle School of Life & Environmental Sciences PO Box 127 Brush Road OURIMBAH NSW 2258

Phone: 02 43494490 Fax: 02 43484145 Email: [email protected]

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.

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ii Foreword

The complementary medicine market provides a real opportunity for alternative cropping by primary producers. There is currently a large consumer demand for natural products that relieve pain and inflammation associated with chronic conditions, and many such products have their origin in traditional plant remedies. One such species is Dodonaea viscosa whose natural distribution range includes Eastern Australia as well as parts of and Oceania.

This project was designed to assess the potential of extracts prepared from this species for application as a topical anti-inflammatory product, and to characterise their active chemical constituents. The project seeks to provide information to enable the manufacture of a product that utilises locally grown raw material.

After comparing levels of active constituents from samples collected in four distinct geographical regions in New South Wales, the Burnbrae site in the Hunter Valley site was selected as the optimal source of germplasm for future cultivation-based projects. were found to contain sufficient levels of flavonoid constituents to account for purported antioxidant and wound healing actions.

While there was no direct evidence of anti-inflammatory activity via an influence on Cox-2 or PGE2, D. viscosa extracts demonstrated significant antioxidant properties, particularly when prepared as hot water decoctions. Initial results also point to an ability to stimulate human dermal fibroblasts and hence enhance wound healing. These findings help to substantiate the traditional use of this species for topical applications, and provide data on which to optimise extraction methods

This project was funded from RIRDC Core Funds which are provided by the Australian Government.

This report, an addition to RIRDC’s diverse range of over 1800 research publications, forms part of our Essential Oils and Plant Extracts R&D program, which aims to facilitate the growth of profitable and sustainable essential oils and natural extracts industries that have commercial potential in Australia.

Most of our publications are available for viewing, downloading or purchasing online through our website:

• downloads at www.rirdc.gov.au/fullreports/index.html • purchases at www.rirdc.gov.au/eshop

Peter O’Brien Managing Director Rural Industries Research and Development Corporation

iii Acknowledgments

I would like to thank my PhD supervisors at the University of Newcastle Dr. Paul Roach and Dr. Mark Lucock, as well as previous supervisors Dr. Douglas Stuart, Professor Ron Wills and Dr. Debbie Shohet. In addition I would like to thank my co-sponsors Southern Cross Herbal Extracts and Penitt Botanicals.

Abbreviations

Cox cyclooxygenase CV Coefficient of variation DAD Diode array detector HPLC High performance liquid chromatography LPS lipopolysacchariide PGE2 Prostaglandin E2 QE Quercetin equivalent TE Trolox equivalent UV ultra violet

iv Contents

Foreword...... iii Acknowledgments...... iv Abbreviations...... iv Contents...... v List of Tables...... vi List of Figures...... vi Executive Summary ...... vii 1. Introduction ...... 1 1.1 Overview ...... 1 1.2 The need for new anti-inflammatory agents...... 1 1.3 Phytochemistry of D. viscosa...... 1 2. Objectives...... 2 3. Methodology ...... 3 3.1 Plant selection, harvesting and processing ...... 3 3.1.1 Harvest location...... 3 3.1.2 Regional variability study ...... 3 3.1.3 Harvesting procedure ...... 3 3.1.4 Drying and processing...... 3 3.1.5 Extraction ...... 3 3.2 Method for separation and identification of chemical constituents...... 4 3.2.1 Analytical equipment ...... 4 3.2.2 Regional variability study ...... 4 3.2.3 Quercetin equivalence method ...... 4 3.3 Bioassays...... 7 3.3.1 Anti-inflammatory activity...... 7 3.3.2 Antioxidant activity...... 8 3.3.3. Wound healing activity ...... 9 3.4 Statistics ...... 10 4. Results ...... 11 4.1 Phytochemistry...... 11 4.1.1 Regional variability study ...... 11 4.1.2 Analysis of D. viscosa and flavonoid standards...... 11 4.1.3 Testing of flavonoid standards ...... 12 4.1.4 Method validation ...... 14 4.1.5 Assessment of quercetin equivalents...... 15 4.1.6 Intra - and inter - specific variation in flavonoids ...... 16 4.1.7 Seasonal comparisons – Burnbrae and Ourimbah...... 17 4.2 Bioassays...... 19 4.2.1. Anti-inflammatory assay ...... 19 4.2.2. Antioxidant assay ...... 19 4.2.3. Wound healing assay...... 23 5. Discussion...... 224 5.1.1 Analysis of chromophores...... 224 5.1.2 Quercetin calibration ...... 224 5.1.3 Environmental factors and flavonoid variability...... 224 5.2 Anti-inflammatory activity...... 225 5.3 Antioxidant assays...... 225 5.3.1 Spectrophotometer assay...... 225 5.3.2 Microplate assay...... 225 5.4 Wound healing assay...... 225 6. Conclusions and Recommendations ...... 226 References ...... 227 Appendix 1. Flavonoids of Dodonaea viscosa ...... 229

List of Tables

Table 1. Sample codes and descriptions ...... 6 Table 2. Retention time and UV data for selected peaks of D. viscosa extracts...... 11 Table 3. Retention times and UV spectra for reference compounds...... 12 Table 4. Quercetin recovery with correction for endogenous component...... 14 Table 5. Revised D. viscosa peaks with UV data, from sample BBX2 ...... 14 Table 6. Comparison of total and individual peaks expressed as QE (μg/mg) ...... 17 Table 7. Antioxidant comparison of two Ourimbah specimens as measured by A. Trolox equivalents and B. % inhibition...... 20 Table 8. Antioxidant activity of Ourimbah aqueous extracts based on concentration...... 21 Table 9. Antioxidant activity of ethanol and aqueous extracts...... 21 Table 10. Comparison of antioxidant activity according to extraction solvent...... 22 Table 11. Levels of antioxidant potency association with solvent selection...... 22 Table 12. Antioxidant activity of hot water decoctions made from fresh (HWG) and dried leaves (HWD)...... 22 Table 13. Antioxidant effects of four D. viscosa specimens. n = number of samples tested...... 23

List of Figures

Figure 1. D. viscosa and ...... 7 Figure 3. Chromophores with UV maxima values for peak #4 and peak #3...... 13 Figure 4. Chromatogram of D. viscosa ethanol extract showing mobile phase gradient...... 13 Figure 5. Chromatogram of BBMLD3 from a Burnbrae male leaf specimen with numbered peaks...... 15 Figure 6. Quercetin equivalents μg/mg D. viscosa leaf of peak #5 for ten mixed samples...... 16 Figure 8. Total measured flavonoids as Q.E.± SEM in Ourimbah and Burnbrae samples...... 18 Figure 9. Season comparisons of Ourimbah specimens for total QE ±SEM ug/mg ...... 18 Figure 10. Seasonal comparisons. Ourimbah and Burnbrae total QE (μg/mg) ± SEM...... 18 Figure 11. Influence of Hopbush extracts on Cox-2 expression (n=1) ...... 19 Figure 12. Mean inhibition ± SEM of 2,2/-azo-bis(2-aminopropane) (AAPH) induced peroxyl radicals by Trolox and D. viscosa ethanol, hot water and cold water extracts...... 19 Figure 13. Mean inhibition ± SEM of AAPH oxidation by aqueous extracts at five concentrations (average of 4 assays)...... 21 Figure 14. Mean inhibition ± SEM of AAPH oxidation by D. viscosa samples...... 23 Figure 15. Difference in Alamar Blue reduction between treated and untreated fibroblasts (average of three tests), sample OB507...... 23

vi Executive Summary

What the report is about This project was designed to assess the potential of extracts prepared from Dodonaea viscosa, an indigenous species previously used in traditional medicine, for application as a topical anti- inflammatory product and to characterise their active chemical constituents. The project seeks to provide information to enable the manufacture of a product that utilises locally grown raw material.

Who is the report targeted at? The success of the project will encourage alternative crops of D. viscosa within the primary producing sector and create an innovative product for the complementary medicine sector.

Background Dodonaea viscosa is an evergreen woody perennial herb found in many parts of the world, most notably in and around the rangeland regions of New South Wales. Ethnopharmacology reporting in Australia and elsewhere indicates a variety of therapeutic uses, in particular as a topical application with anti-inflammatory and analgesic properties. Recent phytochemical studies have confirmed a positive correlation between several groups of active constituents and the traditional usage. Numerous constituents such as triterpenoid saponins and flavonoids have been previously identified, however little work has been conducted on Australian specimens. There is currently a large consumer demand for natural products that relieve pain and inflammation associated with chronic conditions, and this species has the potential to produce a natural extract possessing these characteristics.

Aims/Objectives The main objective of this project was to determine whether extracts of Dodonaea viscosa subspecies angustifolia could provide the biological activity needed for topical formulations to combat common inflammatory disorders such as arthritis and dermatitis.

Methods used An analytical method based on High Performance Liquid Chromatography (HPLC) was designed and validated using the common flavonoid quercetin as a calibration standard, in order to quantify flavonoid levels in a range of D. viscosa samples. Comparisons were made between four distinct geographical regions in New South Wales, and between specimens transplanted from their natural location (Burnbrae) to the Ourimbah campus site with other Burnbrae specimens. Comparisons were also made between different plant sections and different extraction solvents.

Samples were tested for anti-inflammatory activity using Cox-2 and PGE2 assays, for anti-oxidant activity using spectrophotometer and microplate assays, and wound healing activity using a human fibroblast proliferation assay.

Results/Key findings Specimens from Burnbrae, the most accessible of the regional sites, contained slightly superior levels of flavonoids compared to the other harvest sites, hence it was selected as the main harvest site as well as providing genetic stock for cultivation purposes. There was little variation in overall flavonoid levels during the months tested for – however, and capsules had very low levels. Leaves extracted with ethanol provided sufficient flavonoid levels to account for purported therapeutic actions.

While there was no evidence of influence on Cox-2 or PGE2 activity, all D. viscosa extracts possessed antioxidant activity as determined by a peroxyl trapping in vitro method. Hot water extracts showed the most potent antioxidant capacity in both the spectrophotometer and microplate methods, despite containing lower flavonoid levels than ethanol extracts. Initial studies reveal that ethanol extracts of D. viscosa leaves were able to stimulate human dermal fibroblast growth when compared to controls and hence could potentiate wound healing. Aqueous extracts are yet to be tested by this method.

vii Implications for relevant stakeholders On the basis of these results I have confirmed that Australian D. viscosa has sufficient levels of active constituents to account for its reputation as a traditional medicine. It is readily harvested, and extracts prepared using simple techniques have demonstrated antioxidant and wound healing activity in vitro. The constituent levels and biological activity appear to be retained when wild plant stock is domesticated, making it a potential candidate for cultivation, were it to become the basis of a commercialised topical application. These findings help to substantiate the traditional use of this species for topical applications, and provide data on which to optimise extraction methods.

Recommendations 1. Ongoing monitoring for seasonal variation in flavonoid levels at the two sites is required to gain a longer-term perspective and minimise any influence of year-to-year weather fluctuations on the results.

2. Further phytochemical analysis is required to assess presence of non-flavonoid compounds - especially saponins and diterpenes.

3. The lipoxygenase assay using a linoleic acid substrate should be completed in a further attempt to demonstrate a mechanism of anti-inflammatory activity.

4. Continued testing of D. viscosa extracts (including aqueous extracts) in the fibroblast proliferation assay is required, to further substantiate potential wound healing properties, and to establish a concentration – response range.

5. Small field and greenhouse trials could optimise growth conditions and build up stocks in anticipation of future demand. Specimens would be continually monitored for flavonoid levels, as measured by quercetin equivalents.

6. Topical products based on both aqueous and ethanol leaf extracts could be formulated for testing on humans in small clinical studies.

7. The findings of this project could be used as part of an application to have the species considered for listing on the Australian Register of Therapeutic Goods. This will require further financial commitment and approximately 12 – 24 months to acquire toxicology data, paving the way for further work in product formulation.

viii 1. Introduction

1.1 Overview In recent years there has been a renewed interest into the biological activity of traditional plant medicines, and the role of natural products in drug discovery (Butler, 2004). Reasons for this include the great need for new molecular models, as leads into potential new drugs, and for authenticating traditional applications for use in current therapy (Naranjo, 1995). In addition ‘ethno-directed sampling’ of species used in traditional medicine has proven far more fruitful in the identification of new drugs compared to random screening (Graz et al., 2007). With some notable exceptions, Australia has tended to lag behind most of the world in this endeavour. However the native Australian flora could be a potential “goldmine” of biologically active therapeutic substances (Warren, 1990).

Dodonaea viscosa is a locally (New South Wales) common indigenous species, which is easy to harvest and convert to a variety of therapeutic agents. The well-recorded traditional uses in Australia and overseas indicate it is extremely safe – there are no recordings of adverse reactions. However, there are few formal animal toxicological studies (Khalil et al., 2006), nor any clinical trials on humans to formally assert claims of safety and efficacy.

1.2 The need for new anti-inflammatory agents Managing the disability and pain that results from inflammatory diseases is a challenge healthcare practitioners’ face on a daily basis. Along with obvious inflictions such as arthritis and eczema, inflammatory processes are associated with major diseases - diabetes, heart disease and asthma. Besides the accompanying pain and discomfort, prolonged inflammation is destructive to tissues and when sustained, can be an agent in turning a sub-acute or chronic ailment into a degenerative state.

Although arthritis is one of the oldest known diseases affecting a large population of the world, no substantial progress has been made in achieving a cure. Meanwhile the conventional anti- inflammatory drugs such as the COX-2 inhibitors have been found to have significant side effects (Whittle, 2004), while the emergence of a worldwide ‘chemophobia’ against pharmaceutical drugs in general has been well documented (Slikkerveer, 2006). Hence, there is currently a large consumer demand for natural products that relieve pain and inflammation (Bauer, 1999). Similar arguments have been made for the investigation of herbal alternatives to conventional wound healing treatments (Krishnan, 2006, Kumar et al., 2007).

The angustifolia subspecies of D. viscosa was selected because of its local abundance and the fact that extracts and creams made from it by the author and his colleagues have provided clinical evidence of significant anti-inflammatory and analgesic activity (Pengelly, 2003). Considering the potential size of the market for topical anti-inflammatory and wound healing agents, such products would preferable be easily harvested and extracted and come from a renewable source. Despite the natural occurrence of D. viscosa in other parts of the world (Pearman, 2000)eastern Australia is the epicentre of distribution, where relatively large volumes could be harvested from wild stands. The species is widely planted as a hedge and could be readily cultivated in plantations on a wide variety of soil types.

1.3 Phytochemistry of D. viscosa Knowledge of individual chemical constituents of a medicinal plant is essential for optimising extraction procedures, understanding pharmacological activity as well as potential toxicity and interaction with pharmaceutical drugs.

The species in question has been subjected to numerous analytical studies over the last 40 years - however most of these were conducted outside of Australia. Khan, Javed and Khan analysed D. viscosa flowers harvested in India (Khan et al., 1992). In their analysis of flowers harvested in Libya, El-Zwi and Ahme concentrated on the composition of the volatile oil and sterols (El-Zwi and Ahmed 1999).

1

In general the species contains di- and triterpenes, saponins, flavonoids and a complex mixture of other phenolic compounds. It is likely that any therapeutic activity in the herb is associated with polyvalent pharmacological effects brought on by the synergistic combination of several constituents rather than any single isolated one (Wagner, 2005).

Ghisalberti carried out a comprehensive review of the chemistry of the Dodonaea genus, with particular emphasis on D. viscosa. However there are few references to subspecies. He identified 23 flavones from , bark flowers and leaves of D. viscosa, characterised by oxygenation at C-3 and, in almost 50% of cases, methoxylation at C-6 (Ghisalberti, 1998). Siddiqui has also reviewed the chemistry and pharmacology of the species (Siddiqui, 1998). Ghisalberti observed that many uses of the herb by indigenous people from various countries show remarkable similarities, which in turn appear to correlate with the known active constituents (Ghisalberti, 1998).

The major investigation of flavonoids was conducted by Sachdev and Kulshreshtha, who in 1983 isolated eight compounds, providing UV, MS and 1H NMR data for these flavonoids (Sachdev and Kulshreshtha, 1983). Siddiqui’s 1998 review makes reference to eighteen flavonoids including glycosides of quercetin (eg. rutin) and isorhamnetin - these were isolated previously by Nair and Subramanian in 1975 (Siddiqui, 1998). Mata and co-workers isolated sakuranetin, and 6- hydroxykaempferyl-3,7-dimethyl ether from Mexican D.viscosa in 1991. These compounds had not previously been found in D.viscosa (Mata et al., 1991). Leucocyanidins were reported by Sastry and Nayudamma in 1966 (Khan et al., 1992).

More recently Getie et al isolated relatively large concentrations of quercetin, kampferol and isorhamnetin in D. viscosa crude leaf extract from Ethiopia (Getie et al., 2000). A full summary of flavonoids previously identified in D. viscosa and their structures appears in Appendix 1.

The presence of flavonoids or other active constituents at sufficient levels to provide this activity would be a major factor in the choice of harvest location and season, as well as the selection of breeding stock for cultivation programs.

2. Objectives

The main objective of this project was to determine whether extracts of Dodonaea viscosa subspecies angustifolia could provide the biological activity needed for topical formulations to combat common inflammatory disorders such as arthritis and dermatitis.

In particular: (a) To develop analytical techniques to analyse for saponins, flavonoids, coumarins and diterpenes found in D. viscosa subspecies angustifolia. (b) To determine levels of each constituent group in various plant sections and determine the effects of some variables such as growing location (c) To test extracts with a known biochemical matrix for anti-inflammatory properties.

2 3. Methodology

3.1 Plant selection, harvesting and processing 3.1.1 Harvest location Specimens were harvested from ‘Burnbrae’, an organically certified pastoral property in the Liverpool Range region (north-west of Merriwa) of the Upper Hunter Valley. Voucher specimens were authenticated by Dr. Judy West and deposited at the Australian National Herbarium. In 2004 two young seedlings from Burnbrae were transplanted to the University of Newcastle Ourimbah Campus, NSW and planted two metres apart. At the Ourimbah site they have developed into full size specimens and produced flowers and in 2006.

3.1.2 Regional variability study Data on geographical location of specimens held at the National Herbarium, Canberra, were kindly provided by Dr. Judy West – the recognised authority on botany of Dodonaea spp. Details of over 100 specimens collected in New South Wales, S.E. Queensland & N.E. Victoria were provided in spreadsheet form. Entries listed collection date, locality, co-ordinates and habitat description amongst other data.

In order to assess potential variation in the phytochemistry according to geographical location, samples of this subspecies were collected from different geographical zones in New South Wales. From the range of possible locations, four regions were chosen as representing different geographical locations within the state – New England, central-west, southern highlands and south-coast. No specimens were found in the southern highlands site. However samples were able to be collected from Kingston (KG) in New England, Timor (TM) in the central-west and Tilba (TB) on the south coast. Where possible, six samples of 500g leaves (fresh weight) were collected from separate on each site. The fieldwork was conducted during the winter to minimise any deterioration that could result from the delay between harvesting and drying.

3.1.3 Harvesting procedure Leaves and stems were harvested throughout the year, using a combination of long handles shears for the higher branches and secateurs for the lower ones. Leaves from male (BBML), female trees (BBFL) and unknown gender (BBDL) were kept in separate containers. Once harvested, leaves were transferred into Barnel Spring Buckets (Portland USA) in which they were carried back to the drying facility on the day. Flowers and capsules were collected during the late winter and spring. They were stored along with leaves and stems of the same gender, apart from male flowers that contain large quantities of pollen and were therefore kept in separate buckets.

3.1.4 Drying and processing Leaves were spread out on trays and dried in the dehydrator (BBO Forced air system) at 38.50C, ensuring the samples were kept separated. Once dried, plant samples were ground in a Perten Laboratory Mill 3100 (0.8mm sieve) for larger quantities, and by using a mortar and pestle for smaller samples.

3.1.5 Extraction Ethanol extracts 10 mg samples of ground leaves were each added to a clean beaker containing 1mL of 100% absolute ethanol or aqueous-ethanol (1% w/v). The extracts were placed in a water bath sonicator for 5 minutes, after which they were filtered according to the requirements of the experiment being conducted.

3 Aqueous extracts The hot water extraction procedure was based on the traditional herbal decoction method, with concentrations adjusted to meet the standard pharmaceutical definition of 1:20 w/v (50mg/mL) (Cooper, 1972) (Painter, 1998).

A. Prepared from dried leaves Five samples of dried D. viscosa leaves were chopped into small pieces, each weighing 5g. Deionised water (150mL) was poured into Pyrex beakers and brought to the boil on a Tiffany hot plate. The leaves were gently simmered (decocted) for 20 minutes, after which they were strained and the liquid allowed to cool. The solution was then filtered with a Buchner funnel under vacuum, using Avantek #1 filter papers. The volume was measured and adjusted to 100mL by further simmering or addition of deionised water, so that the final volume represents the standard drug/solvent ratio of 50mg/mL. The final decoction was bottled, labelled and stored at -5°C.

B. Prepared from fresh leaves Firstly the moisture content of the fresh leaves was determined. Fresh leaves were harvested and a 5g sample placed in the vacuum oven until dry. The dried sample weighed 1.9g. Therefore, the moisture content was 62%, and the fresh/dried herb weight ratio was 2.7:1.

In order to perform an equivalent procedure to that of the dried leaf decoction (above) 13.5g fresh leaves (5g x 2.7) were chopped up and added to 150mL water and the decoction proceeded as above for dried leaves. The extracts were straw coloured, quite cloudy and frothy, suggesting the presence of saponins.

3.2 Method for separation and identification of chemical constituents

3.2.1 Analytical equipment A Shimadzu LC-10AT HPLC system connected to a Shimadzu SPD-10 Diode Array Detector and a Phenosphere C18, 250 X 4.60 mm/5 µm column (Phenomenex) was used to separate and analyse constituents of D. viscosa. Prior to injection, all samples were passed through a 0.45μg nylon membrane filter into 5ml sample vials, which were then labelled accordingly.

3.2.2 Regional variability study Extracts were prepared from fourteen samples of leaves representing the three sites. Four extracts from Burnbrae samples were also included. Samples extracted in HPLC grade methanol to concentrations of 10mg/mL after which they were subjected to HPLC reverse phase chromatography and diode array detection. Chromatogram peaks were numbered according to UV spectra (wavelength of maximum absorbance) and retention time comparisons (1-10). Peak intensities were equivalent to percentage of absorbance area under the curve (AUC) for individual peak compared with absorbance AUC for 10 main peaks.

3.2.3 Quercetin equivalence method Method development Brolis et al. report a HPLC/ diode array (DAD) method for validation of constituents of Hypericum perforatum, using rutin as external standard (Brolis et al., 1998). The analyte profile of H. perforatum contains several flavonoids including quercetin. Since quercetin is reported to be present in D. viscosa (Getie et al., 2000), and readily available as a pure compound, the method was set up on the Shimadzu system.

The mobile phase solutions were A) Methanol (HPLC grade), B) 50mM aqueous solution phosphoric acid, C) Acetonitrile (HPLC grade). The flow rate was 1mL per min and the mobile phase schedule was set at a gradient flow over 40 min as follows: Starting with 0% A, 85% B, 15% C over 30 minutes the gradient goes to 15% A, 10% B, 75% C. For the next 15 minutes the gradient goes to 15% A, 5%

4 B, 80% C, then for 3 minutes to 0% A, 85% B and 15% C where it stays for 2 minutes. The peaks (including quercetin as reference standard) were detected at 270 nm. All solvents are routinely filtered using an Alltech solvent filtration system with Millipore 0.45um filters.

As the above method was clearly not suitable for separation of aqueous extracts, adjustments were made to the mobile phase to enhance extraction of water-soluble constituents. Both hot and cold water D. viscosa extracts (as described above) were tested, along with the quercetin reference standard. The mobile phase schedule was adjusted as follows: 85% B, 15% C for 30 min, 15% A, 10% B, 75% C for 15 min, 15% A, 5% B, 80% C for 3 min, 0% A, 85% B, 15% C for 2 min.

Reference standards Quercetin, rutin, apigenin, pinocembrin, sakuranetin, fraxetin, kaempferol, isorhamnetin were the selected reference standards. Stock solutions were prepared at concentrations of 0.001g/10ml HPLC methanol.

Method validation - quercetin equivalents Despite the presence of numerous flavonoid-like compounds in the Australian D. viscosa, none of the flavonoid reference compounds appeared to be present. Given the known association of quercetin as a wound-healing agent, and the fact it doesn’t co-elute with any of the peaks on the chromatogram, it was chosen as the calibration standard.

In a move to standardise method validation in chromatography, the International Conference on Harmonization (ICH) have issued guidelines aimed at ensuring methods are reliable, accurate, robust and transferable (Miller, 2005). With this is mind, the following procedures were adopted in order to validate a separation method for flavonoids in D. viscosa.

Demonstration of a linear response using a range of quercetin concentrations In order to utilise quercetin as a calibration standard for D. viscosa leaf chromatography, it was necessary to attain a quercetin concentration curve to test the assumption that there will be a linear response within the concentration range likely to be found in D. viscosa extract.

Quercetin extraction recovery experiment The ratio between the mean peak areas of the spiked samples and the means for low and high quercetin respectively provides the quercetin recovery, however this does not correct for endogenous component. The true quercetin recovery (low and high concentrations) is then calculated by removing contributions from the endogenous component. It is calculated by dividing the means of differences by the mean values for low and high quercetin respectively

Spectrum purity assessment. Using Schimadzu’s ‘post-run analysis’ software function, spectrum window peaks for D. viscosa ethanol extract sample analysed by the revised method were subject to nine-point smoothing and purity assessment. The spectrum window displays the spectra at the upside of the peak, peak top and at the downside of the peak acquired by the peak processing. A purity index is also provided for each peak, in which 1.0 represents a pure spectrum and values approaching 1.0 (eg 0.95-0.99) are considered very close to pure.

Assessment of quercetin equivalents A variety of samples of D. viscosa extracts were prepared from leaves and flowers harvested at Ourimbah and Burnbrae (Table 1). A sample prepared in 2005 (DODO2007) was included in the test, as well as low (15.6ug/mL) and high (250ug/mL) quercetin concentrations. All test samples were prepared at 1% w/v or 10mg/mL. The solvents were 100% absolute ethanol or 45% ethanol. A quercetin concentration curve was generated by plotting the peak areas for the low and high quercetin samples (mean of two injections), plus a blank for the corresponding retention time. Spectrum purity

5 assessment was conducted on all ten peaks from two representative chromatograms (OUR806 and BBFL09). Spectra were analysed in the range of 250-400mm.

Table 1. Sample codes and descriptions Code Description OUR806 Ourimbah specimen leaf, harvested August 2006 OUR906 Ourimbah specimen leaf, harvested September 2006 OUR45 Specimen OUR906 extracted with 45% ethanol DODO1107 Ourimbah specimen leaf, harvested July 2005 BBFL07 Burnbrae leaf, female specimen harvested July 2006 BBFL09 Burnbrae leaf, female specimen harvested September 2006 BBFL45 Burnbrae female leaf, extracted with 45% ethanol BBMF Burnbrae male BBML Burnbrae leaf, male specimen BBMF45 Burnbrae male flower, extracted in 45% ethanol

Individual peak areas Peak #5 was the dominant peak in the chromatograms of most of the samples, hence it was chosen for this study. QE values for each sample were calculated as described above.

Total measured flavonoids (sum of 18 peaks) Total flavonoids were calculated by adding up the peak areas (means of two injections) for all peaks. The resultant value cannot measure the exact level of flavonoids as there may be smaller peaks that may represent flavonoids or flavonoid metabolites, however these would represent a relatively small percentage of overall peak areas. The total peak areas were substituted as the ‘y’ value in the regression equation

Individual compounds (peaks) as a percentage of overall flavonoids This was calculated as a simple ratio between the relative peak areas (Mv/sec) of peak #5 and total flavonoids (peaks #1-18)

Intra- and interspecific variation of flavonoids in two plant specimens This experiment was designed to assess possible variation in flavonoid levels between the two specimens cultivated on the Ourimbah campus (two metres apart), along with possible variation within the two specimens.

Leaves of the two D. viscosa specimens (L and R) were harvested so that ten separate leaf samples of each were obtained. Specimen R was displaying an abundance of capsules at the time, while specimen L had a sparse covering of capsules. Once dried, the leaves were ground and extracted with absolute ethanol as described previously to a concentration of 10mg/mL. Sample vials were prepared by injecting through 0.45μg syringe filters. Samples were labelled A1-A10 and B1-B10. Solvent blanks consisted of absolute ethanol. Low and high quercetin concentrations are included as calibration standards.

By plotting the quercetin peak areas for 0, 15.6 and 250μg/mL a linear concentration gradient was established. For each sample the means of two injections were plotted on an excel spreadsheet for all peaks. Quercetin equivalents were calculated by substituting the peak areas for each sample, as nanograms on column, which were then converted to μg of D. viscosa leaf per mL of solvent. Coefficient of variation (CV) was calculated for each peak.

Seasonal comparisons – Burnbrae & Ourimbah Mean peak areas were established for eighteen peaks, which were converted to QEs using the regression equation and expressed in μg/mg leaf, with standard deviations and standard error of the mean. Unpaired T-tests were applied to assess variability between corresponding compounds based on QE values.

6

Figure 1. D. viscosa leaf and capsule

3.3 Bioassays 3.3.1 Anti-inflammatory activity These studies were conducted under contract by the Natural Products Pharmacology Unit, Southern Cross University under the direction of Dr. Lesley Stevenson, using samples provided by the author.

100g each of three batches of D. viscosa were selected as follows: 1. BB12 (Burnbrae sample) 2. B2 (Timor sample) 3. B3 (Tilba sample) The dried samples were stored at room temperature until ground and extracted. The samples were ground in a Retsch SM-200 grinder and extracted in 100% water, methanol, or acetone. Samples were sonicated for 5 minutes and allowed to stand at room temperature overnight. The extraction solvents were then poured from the ground tissue, and the weight of the extracts determined after freeze- drying.

Cox-II assay The samples were tested in vitro for their ability to inhibit Cox-2 expression post lipopolysaccharide (LPS) stimulation in whole blood monocytes. The extracts were all at 10mg/mL concentration.

Aliquots of human fresh whole blood were preincubated with each sample for two hours at 37°C. (LPS) was then added to each aliquot (except unstimulated controls) for a final LPS concentration of 0.01μg/mL and the aliquot incubated for a further two hours at 37°C. Samples from each aliquot were then stained with CD14 (monocyte marker) monoclonal antibody (mAb) then subsequently stained intracellularly with Cox-2 mAb. The percentage of monocytes expressing Cox-2 was then determined by flow cytometry using a Becton Dickinson FACSCalibur instrument. Unstimulated, LPS stimulated and dexamethasone (positive inhibitor) controls were also run. The percentage activation of Cox-2 for each extract was determined using the respective solvent control value as a reference.

Prostaglandin E2 Inhibition (PGE2) Swiss Albino fibroblast cells 3T3 were plated out (1 × 105 cells/mL, 100μL/well) into 96-well tissue culture plates. The cells were grown overnight at 37 °C in 5% CO2 atmosphere. The D. viscosa samples, as prepared above, were tested at a final concentration of10μg/mL and 1μg/mL. Samples were added to the cells (5μL/well), and the plates incubated (37 °C, 3h) before addition of calcium ionophore A23187 and further incubation (20 min). The positive control was aspirin (100μM, equivalent to 18μg/mL). Solvent and media controls, with and without calcium ionophore A23187, were included.

7 The plate was then centrifuged (1000 RCF, 5 min) and the culture supernatants were removed and stored at –80 °C, until they were diluted by serial dilution 1:500 in EIA assay buffer. The supernatants were assayed for prostaglandin E2 using a monoclonal antibody enzyme-linked immuno-absorbant (EIA) assay kit, according to the kit protocol (Cayman Chemical).

3.3.2 Antioxidant activity Assay for spectrophotometer This assay is based on a method developed by Dunlap et al. (2003) for determining the peroxyl- trapping activity of extracts derived from marine organisms using a reduced xanthene leuco-dye dihydrorhodamine (DHR) as substrate for oxidation by peroxyl radicals generated by 2,2/- azo-bis(2- aminopropane) dihydrochloride (AAPH) (Dunlap et al., 2003).

The following reagents were prepared: 0.1mM Tris-HCl obtained from Sigma (St. Louis, MO, USA) (pH7.4) adjusted to pH 7.4 using 10mM of sodium hydroxide solution; Ethylenediaminetetra-acetic acid (EDTA) 10mM in D.I. H20. EDTA Sigma E-4884, 250μm DHR solution (dihydrorhodamine-123 – Sigma D1054) - consisting of 0.0017g of DHR powder, 12mL HPLC grade methanol; 125μL of 10mM EDTA and 7.875mL Tris-HCl 0.1mM; 50mM AAPH 2,2/- azo-bis(2-aminopropane) dihydrochloride – Wako 017-11062; Lot KLJ4720) obtained from Wako Pure Chemical Industries (Osaka, Japan). 200μM Trolox (6-hydroxy- 2,5,7,8-tetramethylchroman-2-carboxylix acid) – Sigma CAS 53188-07-1.

Different concentrations of D. viscosa samples and controls were tested in cuvettes, adding D.I. water to make up to 225μL (see Table 1). Then 500μL DHR solution, 25μL of Tris-HCl and 500μL AAPH were added to each cuvette.

Oxidation kinetics was monitored on a Varian Cary 50 UV Visible spectrophotometer, on which the following parameters were incorporated in the application settings: Constant temperature 260C, wavelength 501 nm, cycle time 5 minutes, end time 60 minutes

Following the method by Dunlap, antioxidant activities are expressed as percentage of inhibition of the control rate of oxidation (ie. water). ⎡ inhibition rate of extract ⎤ oxidation % oxidation inhibition = 100 -1 ⎣⎢ control rate of oxidation ⎦⎥ where inhibition rate is equivalent to the absorbance slope of the sample and control rate of oxidation is equivalent to the absorbance slope of water.

Ethanol, hot- and cold-water extracts were prepared in the usual way, and adjusted to concentrations of 10mg/mL (1% w/v), 20mg/mL (2%) and 40mg/mL (4%). In order to assess variability of peroxide inhibition by different aqueous ethanol extracts, a second assay was set up, in which the samples consisted of 30, 45 and 100% ethanol extracts, at concentrations of 10 and 40mg/mL.

Assay for microplate Dunlap’s previously described spectrophotometric method can also be conducted on a 96-well microplate reader once the sample and reagent volumes are scaled down (Dunlap, 2003). This method allows five times the number of samples per run, easier transfer of data to spreadsheets and saves on the quantity of reagents in use. Trolox dilutions commonly employed are 0, 37.5, 75, 150 and 300uM.

Sample volumes and numbers are scaled according to Dunlap’s alternative assay (Dunlap, 2003)- the revised reagent volumes (all in μL) are: methanol 20, sample, Trolox dilution or blank 40, DHR 30, AAPH 60 for a total of 150μL per well. Absorbance is read on a micro-plate reader (Benchmark plus, Bio-Rad, Sydney).

8 Extraction solvent comparisons In order to further explore the relationship between extraction solvent and activity, an assay was set up using samples extracted with a range of aqueous/ethanol solvents. These were prepared from freshly harvested OURL leaves using the following solvents: 100% absolute ethanol, 60% ethanol, 45% ethanol, 25% ethanol. The samples (four for each solvent) were extracted to a uniform concentration of 10mg/mL. The aqueous extract was also compared.

3.3.3. Wound healing activity Fibroblast proliferation assay The purpose of this procedure is to investigate the potential of leaf extracts of D. viscosa to stimulate the growth of fibroblasts, using a colorimetric assay.

Culturing of fibroblasts Cryogenically preserved human dermal fibroblasts (HDFa) were thawed and transferred to 25cm2 tissue culture flasks in a solution of supplemented growth media. The number of viable cells per ml was determined with a haemacytometer and an inverse microscope (Leika DMIL) from a 20ul sample combined with trypan blue solution. The culture was incubated in a 370C, 5% CO2/95% air humidified cell culture incubator (Sanyo MCO-20AIC). After 24hours fibroblasts adhered to the bottom of the flask (monolayer) and formed characteristic shapes. The growth medium was replaced every second day until the fibroblasts approached confluence. As the culture neared confluence (70- 90%), the subculturing procedure was adopted.

Addition of trypsin/EDTA solution causes cells to revert to immature round form, while addition of trypsin neutraliser terminates the procedure. Cells in the neutraliser solution were centrifuged for seven mins at 200 C (Sigma 4K15) after which the supernatant solution was removed. The cell pellet was resuspended in supplemented growth media, from which new culture flasks were established. In the explant procedure sub-cultured fibroblast solution was dispensed from centrifugal tubes to each well of a 96-well flat-bottomed microplate in preparation for the assay. Cells were vortexed regularly for even distribution during the seeding procedure. When seeding was complete, the microplate was incubated for 24 hours.

Experimental design Alamar Blue is a redox inhibitor that exhibits colorimetric and fluorescence changes upon exposure to cellular metabolic reduction. Changes in cell numbers were measured either by spectrophotometric or phosphorescent detectors. Alamar Blue was chosen over tetrazolium salts (eg MTT) in being harmless to cells and readily water-soluble.

A 10% Alamar Blue solution, serial dilutions of sample extracts and set amounts of supplemented culture media were added to each well. Some wells were reserved as positive, negative and blank controls. To assess the proliferative effects of D. viscosa extracts on human fibroblasts, cells grown to near confluence were combined with different concentrations of D. viscosa extracts. The control blanks consisted of normal cell culture without addition of D. viscosa extracts.

Cell proliferation induces chemical reduction of the media, resulting in change of colour from blue to red, and is determined by measuring absorbance at 570nm and 600nm. Percent reduction was determined using an equation and molar extinction coefficient values for oxidised and reduced Alamar Blue at the two wavelengths as follows (Bioscource, 2005). ε ε % Reduced = ( ox)λ2 Aλ1- ( ox) λ1 Aλ2 X 100 ε ’ ε ’ (red)λ1 A λ2 - ( red) λ2 A λ1 ε ( ox) = molar extinction co-efficient of Alamar blue oxidised form ε ( red) = molar extinction co-efficient of Alamar blue reduced form

λ1 = 570nm

9 λ2 = 600nm A = absorbance of test wells A’= absorbance of negative control (blank containing media + Alamar Blue but no cells)

When a positive control is used, the percentage difference in reduction between treated and untreated cells is equivalent to the degree of proliferation.

When the cell cultures are inspected by microscope after incubating overnight, contents of treated wells and positive blanks have turned from blue to pink. Negative and blank control wells are colourless or blue.

Assessment of cell proliferation Reduction of Alamar Blue occurred in all samples at all dilutions. In nearly every case complete reduction (100%) was achieved within 48 hours, hence the assay was not prolonged beyond this point. There were so significant differences between different samples or their concentrations. The high relative percentage reduction in the first two hours of the assay raised the question as to whether the same extracts may induce Alamar Blue reduction by inherent antioxidant properties (D. viscosa contains flavonoids and other compounds linked to antioxidant activity), or by the activity of mitochondrial enzymes (O'Brien et al., 2000).

In order to test this hypothesis, a microplate was set up so that four dilutions of six extracts were added to the Alamar Blue solution and supplemented culture media in the absence of fibroblasts. The plate was incubated and scanned at 0, 2, 4 and 24hours. Absorbance values showed no demonstrable difference between the sample extracts and control (solvent) blanks.

Comparison of proliferation rates in treated and untreated fibroblasts Since cell proliferation is indicated by Alamar Blue reduction, actual proliferation rates may be determined by comparing reduction of treated fibroblasts with that of untreated controls (ie solvent blanks). The percentage difference in reduction is determined by an equation incorporating the molar extinction value for oxidation of Alamar Blue

3.4 Statistics Wherever possible analyses were carried out with five or more samples and their means reported. Standard deviations (SD), standard errors (SEM) and coefficients of variation (CV) are calculated to monitor the consistency of the results. Data collected was subject to students T-test (two data sets) or one way ANOVA for three or more data sets (p <0.05). The Tukey-Kramer HSD test for multiple comparisons at the 5% significance level is used to test the differences between all pairs of means

10 4. Results

4.1 Phytochemistry 4.1.1 Regional variability study All chromatograms provided ten well-defined peaks, most of which had UV spectra typical of flavonoids. When means for the total peak areas for each site were compared, the highest levels were recorded by the Burnbrae samples – these were almost double those recorded in the Timor and Tilba samples (Figure 2). Application of the Tukey-Kramer HSD test to mean total peak areas confirmed Burnbrae had significant variation while the other three sites did not.

Analysis of peak areas with one-way ANOVA reveals significantly different intensities for several peaks (data not shown). For example: Peak #1 is consistently higher at Timor and Burnbrae, peak #2 is significantly higher at Kingston and Burnbrae relative to other sites.

A 70000 60000 B 50000 B B 40000 30000 20000

(thousands) 10 0 0 0 0 Totalof area peaks 10 BB TM TB KG

Figure 2. Mean total peak areas ± SEM for four sites. Key: A– significantly different (p<0.05) B- not significantly different BB – Burnbrae, TM – Timor, TB – Tilba, KG – Kingston.

4.1.2 Analysis of D. viscosa and flavonoid standards The major peaks for the D. viscosa ethanol extract all eluted in the 26-36 minute region, however most peaks in the aqueous extracts eluted between six and eighteen minutes. The hot water extract displayed a second series of peaks in the 26-minute region, although this group of peaks did not appear in the cold-water extract chromatogram. Retention times and UV data for rutin and quercetin did not correspond with any of the D. viscosa peaks (Table 2).

This method gave satisfactory separation of peaks in D. viscosa ethanol extracts and less satisfactory results for aqueous extracts, presumably due to the presence of significant levels of aqueous constituents.

Table 2. Retention time and UV data for selected peaks of D. viscosa extracts Peak description Retention time Wavelengths of maximum (mins) absorbance Rutin standard 14.86 274, 340 Quercetin standard 29.6 257, 375 Dodonaea ethanol extract First main peak 28.8 218S, 277, 340 peak 30 257, 375 largest peak 31.4 269, 346 second largest peak 33.2 272, 340 Dodonaea hot water extract Very large peak 6.5 217, 326, 237S, 297S Large peak 10 317 Peak 13.6 254, 354, 268S Peak 33.9 269, 345

11 Peak description Retention time Wavelengths of maximum (mins) absorbance Dodonaea cold water extract Very large peak 6.6 217, 326, 237S, 297S Peak 7.8 228, 310 Peak 10.2 217, 326, 237S, 297S

Revised method Given the compounds had virtually all eluted in the first half of the gradient run, it was decided to reduce the run time from 65 to 30 minutes, adjusting the gradient accordingly. In addition to quercetin, apigenin was used as an external standard, since some chromophores from D. viscosa extracts in the previous experiment matched closely the UV data reported for apigenin and some of its glycosides.

The D. viscosa extracts were prepared as previously described, to final concentrations of 10mg/mL. Other samples tested were quercetin, D. viscosa ethanol extract and hot water extracts. Quercetin and apigenin eluted at 11.1 minutes and 12.6 minutes respectively. The UV for apigenin was 267, 335. Good peak separation was achieved for the D. viscosa ethanol extract, with 9-10 distinct peaks and the major peak at 13.7 minutes, with an apparent new peak appearing at 26.4 minutes with a UV reading of 274, 340 – typical of 3-OH substituted flavonols (Markham, 1982).

4.1.3 Testing of flavonoid standards Excellent peak separation was achieved for the D. viscosa extract in line with the previous result. There were eight major peaks, the largest eluting at 13.7 minutes. Retention times and UV data of D. viscosa peaks were compared with those of the standards

Table 3. Retention times and UV spectra for reference compounds Flavonoid R.T. (mins) U.V. spectra Rutin 6.48 274, 340 Quercetin 11.05 272, 340 Apigenin 12.66 275, 338 Pinocembrin 16.68 207, 211, 290 Sakuranetin 16.4 217, 286 Fraxetin 6.5 271, 337

None of the standards tested match the peaks in the D. viscosa extract by both retention times and UV spectra. The UV data suggest most of the peaks flavones and flavonols, but these are yet to be identified.

Peak purity assessment While most of the peaks appear to be pure or near pure, there was some tailing observed for peak #3 at 13.2 minutes, and in peak #4 there appear to be two compounds eluting together. It was concluded that peak separation could be improved by extending the middle range of the solvent gradient, thereby increasing the run time to 40 minutes.

12 D. viscosa ethanol extract (DOD906) was analysed using this method. Overall separation is much improved, and peak # 4 appeared to be a pure chromophore (Figure 3a). The tail from peak #3 was isolated and the peak then also appeared as a pure chromophore (Figure 3b). UV data suggested most or all of the 9-10 peaks represent flavonoids. This gradient was retained as the standard method (file DODETOH4) for analysis of D. viscosa ethanol extracts (Figure 4).

Figure 3. Chromophores with UV maxima values for peak #4 and peak #3.

4

3

Figure 4. Chromatogram of D. viscosa ethanol extract showing mobile phase gradient. Numbers represent peaks #3 and 4. Full line – aqueous, dotted line – acetonitrile, methanol gradient not shown.

13 4.1.4 Method validation Quercetin Concentration Study This concentration relationship provides the basis for linear regression, with the equation: y = 2.212.52x – 339759. Number of samples (n) = 7, Correlation co-efficient (r2)= 0.9962, P = <0.0001 (slope). The linear relationship was consistent if the zero concentration value was added: r2 = 0.9961

Quercetin extraction recovery experiment This test confirmed the total absence of quercetin in the D. viscosa samples and the presence of small quantities of an unknown compound co-eluting with quercetin in the spiked samples. It also validated the methodology in that full recovery of quercetin is attained.

Table 4. Quercetin recovery with correction for endogenous component Conc. Mean of Standard Recovery Standard CV n ug/mL differences deviation % deviation % Low quercetin 15.6 760833 105389 121 3.3 2.4 10 concentration High quercetin 250 11563812 343094 107 2.8 2.5 10 concentration

Quercetin calibration On the basis of the retention times ten main peaks were confirmed as present in all specimens tested. In addition to these ten peaks, extra peaks with flavonoid UV characteristics were observed. All were tested for peak purity (as described above) and thereafter included in the quantification of total flavonoids. Adoption of these changes led to a revised total of 18 peaks, although not all peaks are present for all specimens – full details are listed in table 5. In addition, a representative chromatogram with numbered peaks is shown in figure 5.

Table 5. Revised D. viscosa peaks with UV data, from sample BBX2 Peak no. Time (mins.) UV (λmax in UV(λmax UV Purity spectrum Mμ) in Mμ) Up Down 1 11.8 278 340 .9997 .9941 2 12.5 261 340 .9936 .9889 3 13.5 270 350 .9956 .9924 4 15.5 266 335 .9212 .9802 5 16.5 270 340 .9925 .9984 6 16.9 270s 350 .9901 .9583 7 17.3 269 346 .9954 .9999 8 18.2 261 333 .9640 .8802 9 19.5 271 346 .8948 1.0 10 19.8 269 342 .9959 .9994 11 20.2 270 346 .9992 .9997 12 22.1 271 343 .9927 .9996 13 23.1 271 340 .9981 .9982 14 25 272 342 .9997 .9999 15 25.9 272 341 .9947 .9999 16 27.5 272 340 .9998 .9999 17 32.7 270 340 .9933 .9998 18 35.2 272 340 .9998 .9998

14 5 7 9 11 13 14 6 8 12 2 3 10 4 1 15 16 17 18

Figure 5. Chromatogram of BBMLD3 from a Burnbrae male leaf specimen with numbered peaks.

4.1.5 Assessment of quercetin equivalents Individual peak areas Ourimbah samples contained higher QE levels of peak #5 relative to the Burnbrae samples, and within the Burnbrae samples the male flowers contained lower levels compared to the leaves (see Table 6). This demonstrated a high level of variability between the samples (CV = 81).

Total Flavonoids These results reveal less variability (CV=45) compared to peak #5 only (CV = 81). Samples extracted with 45% ethanol have lower flavonoid levels compared to those extracted with 100% ethanol, while the male flowers contain less than the leaves as was the case in the measurement of peak #5 only (Figure 7).

Levels of individual compounds as percentage of total measured flavonoids For Ourimbah samples, peak #5 consisted between 48-50% of total flavonoids which was much higher than for Burnbrae (12-16%) – data not shown.

15 Sample QE ug/mg A 22.1 25.0 B 14.3

20.0 C 12.4 D 19.1 15 . 0 E 3.9 (ug/ml) (ug/ml) F 7.6 10 . 0 G 3.8

Quercetin equivalents 5.0 H 2.2 I 3.2 0.0 ABCDEFGH I J J 2.1 Samples Mean 9.1

Key: (A) OUR806 Ourimbah leaf; (B) OUR906 Ourimbah leaf; (C) OUR45 Ourimbah leaf, 45% et hanol; Stdev 7.4 (D) DODO1107-Ourimbah leaf; (E) BBFL07- Burnbrae leaf , female; (F) BBFL09 Burnbrae leaf , female ; (G)BB FL45 B urnbrae leaf , f emale45% ethanol; (H) BBMF- Burnbrae male flower; (I)-BBML-Burnbrae Sem 2.2 male leaf; (J)BBM F45 Burnbrae male flower, 45% et hanol CV 81.7 Figure 6. Quercetin equivalents μg/mg D. viscosa leaf of peak #5 for ten mixed samples. See table 1 for sample details

60 Sample QE ug/mg

50 OUR806 46.4

40 OUR906 29.6 OUR45 25.0 30 DODO1107 38.8 20

(ug/mg leaf) BBFL07 27.5 10 BBFL09 48.9 Quercetin equivalents 0 BBFL45 26.2 ABCDEFGHI J BBMF 15.4 Samples Key: (A) OUR806 Ourimbah leaf ; (B) OUR906 Ourimbah leaf ; (C) OUR45 Ourimbah leaf , 45% Etoh; (D) DODO1107-Ourimbah BBML 27.3 leaf ; (E) BBFL07- Burnbrae leaf , f emale; (F) BBFL09 Burnbrae leaf , f emale ; (G)BBFL45 Burnbrae leaf , f emale 45% Etoh; BBMF45 14.0 (H) BBMF- Burnbrae male f lower; (I)-BBML-Burnbrae male leaf ; (J)BBMF45 Burnbrae male f lower, 45% Etoh Mean 29.92 Figure 7. Quercetin equivalents μg/mg D. viscosa leaf for sum of all Stdev 11.66 peaks. See table 1 for sample details. SEM 3.53 CV 39.0

4.1.6 Intra - and inter - specific variation in flavonoids Table 6 reveals much higher total QE values for specimen L (35.85 μg/mL) compared to specimen R (16.7 μg/mL), indicating higher overall flavonoid levels. In order to test for inter-variability between corresponding peaks, an unpaired t-test was conducted on the QE. of each compound. Twelve peaks indicated highly significant differences (P = <0.01), while there were no signs of differences for peaks #11, 12, 15 and 16 (P = >0.05).

Individual compounds as percentage of overall flavonoids This was calculated as the ratio between the peak areas of each compound for the two specimens relative to the total peak area for each sample, as measure by absorbance rates (Mv/sec) or QE values (Table 6). Despite notable differences in peaks # 5, 7, 9, 10 and 14 there is no significance overall between the two sets of mean percentages of total measured flavonoids (paired t-test P = 0.99) whether calculated on absorbance or QE data.

16 Table 6. Comparison of total and individual peaks expressed as QE (μg/mg)

Specimen Specimen L n = 10 R n = 9 Quercetin equivalents Peak # Means % total Means % total 1 0.96 2.40 0.52 2.56 2 1.02 2.54 0.54 2.73 3 2.77 7.81 1.22 7.45 4 1.09 2.79 0.70 3.83 5 15.90 46.99 3.17 21.08 6 1.86 5.09 0.80 4.50 7 0.00 0.00 0.78 4.40 8 0.00 0.00 0.00 0.00 9 0.59 1.30 1.52 9.51 10 4.29 12.35 0.74 4.15 11 1.24 3.24 1.13 6.79 12 1.54 4.15 1.41 8.73 13 1.00 2.53 0.37 1.47 14 0.94 2.34 1.67 10.79 15 0.66 1.49 0.64 3.42 16 1.30 3.41 1.11 6.65 17 0.00 0.00 0.00 0.00 18 0.68 1.57 0.47 2.17 Total s 35.85 100.00 16.7 100.24 Stdev 4.19 2.74 SEM 1.27 0.91 CV 11.69 16.36

4.1.7 Seasonal comparisons – Burnbrae and Ourimbah Comparison of leaves and capsules – October 2006 Ourimbah L specimens had the highest total level of flavonoids (QE35.7), double the next highest - Burnbrae male leaf (QE18.4) - as shown on Figure 7. Extracts derived from capsules had quite low levels by comparison (QE BBFC 7.6, OURC 4.37). There was high variability (CV=71) between the six specimens.

As a means of establishing which of the peaks are responsible for the variation in overall total measured flavonoids, unpaired T-tests conducted on QE values for each peak showed significant variation in all samples (6 out of 6 tests) for peak #6 and in 5 out of 6 samples for peak #13, while in peaks #2 and 17 only 2 out of 6 samples were variable.

17 40 35 30 25

20 leaf 15 10 5 Ug quercetin equivalents/mg 0 OURL OURR OURC BBML BBFL BBFC Sam ples

Figure 8. Total measured flavonoids as Q.E.± SEM in Ourimbah and Burnbrae samples. Values are means of 5 samples for each specimen

Comparison of Ourimbah and Burnbrae specimens Oct 2006 – April 2007 Of the two Ourimbah specimens, OURL consistently produced higher levels of overall flavonoids (as measured by QEs) (Figure 9). Due to the more infrequent harvesting of Burnbrae specimens, these comparisons were made on three occasions between October 2006 and February 2007. In each case the Ourimbah L specimen had the highest QE level followed by the male Burnbrae specimen (Figure 10).

50 45 40 35 30 25 20 15 10

Quercetin equivalents ug/ml 5 0 Oct Oct B Dec Jan Feb Mar Apr/May OURL OURR Figure 9. Season comparisons of Ourimbah specimens for total QE ±SEM ug/mg

60 50 40 30 20 10 Total Q.E.sTotal (ug/ml) 0 Oct Dec Feb OURL OURR BBFL BBML

Figure 10. Seasonal comparisons. Ourimbah and Burnbrae total QE (μg/mg) ± SEM

18 4.2 Bioassays

4.2.1. Anti-inflammatory assay Cox-2 expression None of the extracts showed inhibition of Cox-2. The water extracts showed marked activation of Cox-2, with extracts from leaves B2 and BB12 showing considerable increases in the absence of LPS (40% & 9% respectively) – Figure 11. 60 40 20 0 -20 ABCDEFGHIJ -40 % of controls -60 -80

Key:A-DMS cont rol; B-B2(Acet one); C-BB12(Acet one); D-C2(Acet one); E-B2(Methanol); F- BB12(Methanol); G-B3(Met hanol); H-B2(Water); I-BB12(Water); J-B3(Water).

Figure 11. Influence of Hopbush extracts on Cox-2 expression (n=1)

PGE2 Inhibition The water and methanol extracts at a concentration of 1mg/mL slightly inhibited the production of PGE2 in cells. However, the level of inhibition detected was very slight, and falls within expected biological variation.

4.2.2. Antioxidant assay Spectrophotometer method Ethanol extracts The Trolox extracts demonstrated a linear concentration response (R2 = 0.96) in the first assay for D. viscosa leaf extracts. With the exception of the ethanol extract ET501, 0.5% extract (mean inhibition 18%) all D. viscosa extracts demonstrated levels of inhibition equivalent to or higher than the Trolox concentrations in this assay. Hot water extracts showed 95% inhibition at 4%, 92% inhibition at 2% and 79% inhibition at 1%. The other notable result came from the 4% ethanol extract, which produced a mean inhibition rate of 96% (Figure 12).

120

100

80

60

40 Mean inhibition % inhibition Mean 20

0

r e 0 % % 30 6 1 2 2% 4% at ox W l th th W W o olox rolox90 E E Eth 4% HW 1% H H CW 1%CW 2%CW 4% Tr Tr T Extract type

Figure 12. Mean inhibition ± SEM of 2,2/-azo-bis(2-aminopropane) (AAPH) induced peroxyl radicals by Trolox and D. viscosa ethanol, hot water and cold water extracts.

In the second assay series, Trolox extracts once again demonstrated a concentration dependent inhibition, varying from 16% for Trolox 30μM to 54% for Trolox 90μM. All extracts demonstrated

19 mean levels of inhibition equivalent to or higher than the effect of these Trolox concentrations. The O3A sample (4% extract/100% ethanol) showed the highest mean inhibition value (95%), a result almost identical with the first assay series result. The 30% ethanol samples (O1,O1A) showed similar rates of inhibition at both concentrations.

Microplate method Antioxidant assay for microplate Antioxidant effects of D. viscosa ethanol extracts were monitored over a 12-month period, for Ourimbah and Burnbrae leaf specimens. Percentage of inhibition was determined by comparison between mean absorbance of samples and blank controls. Trolox curves were established by using the log10 concentration value, thereby achieving a curvi-linear dose response for each assay. By inserting the antilog of this value in the regression equation, Trolox equivalents (TE) in mMols were established, which were then converted to mg equivalents.

Sample assay During March 2007, two assays were performed on Ourimbah L and R specimens (10mg/mL ethanol extracts). Typical curvi-linear Trolox curves were established (R2 = 0.96, 0.98). The results (Table 7) show that OURL was more potent in both assays.

Table 7. Antioxidant comparison of two Ourimbah specimens as measured by A. Trolox equivalents and B. % inhibition.

Date Sample Kinetics Log10x TE (mM) TE (mg) A. 23 OURL 4.46 2.02 104.7 10.47 OURR 4.92 1.8 63.09 6.31 27 OURL 2.48 2.72 524.8 52.48 OURR 3.78 2.16 144.54 14.45

Sample %Inhibition St.dev. CV SEM B. 23 OURL 48.74 2.37 4.8 0.75 OURR 43.51 7.39 14.96 2.34 27 OURL 69.44 4.44 5.62 1.41 OURR 53.33 4.82 10.14 1.52

During August 2007 Ourimbah ethanol extracts were prepared to reflect different concentrations. Serial dilutions from the stock solution of 10mg/mL provided a range of concentrations down to 0.625 mg/mL. Trolox curvi-linear response relationships were established in all assays, and inhibition rates and TE values calculated as described above. Little variation in inhibition and TE values occurs as a result of concentration, while in some cases the lowest concentrations provided the highest antioxidant levels. Response curves created using log 10 sample concentrations values did not show significant correlation (r2 = 0.63).

Aqueous extracts The procedure and sample concentrations outlines above were repeated using previously prepared aqueous (hot water) extracts from OURL. In the first assay a Trolox curvi-linear response was established (r2 = 0.96). The highest inhibition rate (82%) was recorded for the 5mg/mL samples, while all results tended to be higher than for previous assays with ethanol extracts. A curvi-linear relationship also occurred between sample concentration and inhibition (r2 = 0.91), which became 0.985 if the highest and lowest concentrations were omitted.

20 Table 8. Antioxidant activity of Ourimbah aqueous extracts based on concentration Sample mg/ml TE mg %Inhibition stdev AQ10 74.13 80.81 <0.01 AQ5 83.18 82.04 0.04 AQ2.5 61.66 78.35 0.05 AQ1.25 34.67 71.22 0.45 AQ.62 23.99 66.79 0.08 AQ.31 22.38 65.81 0.20

Three subsequent assays were performed and the results summarised for inhibition and TE (Figure 13). There was some inconsistency in these results as demonstrated by the error bars. Regression analysis performed on log10 sample concentration showed a slight trend towards a curvi-linear response (R2 = 0.74).

100

90

80

70

% inhibition % 60

50 0.62 1.25 2.5 5 10 Concentration (mg/ml)

Figure 13. Mean inhibition ± SEM of AAPH oxidation by aqueous extracts at five concentrations (average of 4 assays)

Extraction solvent comparisons Ourimbah L and R leaf specimens extracted by 100% ethanol were compared with the previously prepared aqueous extracts at concentrations of 10 and 5mg/mL. The aqueous extracts were clearly the most potent, particularly when comparing TE values (Table 9).

Table 9. Antioxidant activity of ethanol and aqueous extracts Sample TE mg %Inhibition stdev Std err CV L10 2.69 48.55 0.54 0.16 19.14 L5 2.51 47.64 0.56 0.17 19.56 R10 3.31 50.73 0.27 0.08 9.91 R5 2.09 45.64 0.25 0.07 8.26 AQ10 37.15 77.64 0.06 0.03 4.68 AQ5 63.10 83.64 0.10 0.06 11.11

Trolox concentrations produced the curvi-linear response (R2 = 0.99). The aqueous extract was again the most potent (79% inhibition). High and low percentage ethanol extracts were more potent than the middle range (45, 60% ethanol) – see Table 10. Due to these slightly contradictory results the test was repeated but without the aqueous extract – whose potency was by now well established – and the results were very similar.

21 Table 10. Comparison of antioxidant activity according to extraction solvent. % ethanol TE mg %Inhibition stdev Std err CV 0 36.31 79.05 0.04 0.01 2.92 25 10.00 56.71 0.50 0.18 20.17 45 2.69 33.68 0.57 0.20 14.90 60 1.86 27.27 0.51 0.18 12.14 100 9.33 55.67 0.49 0.17 19.00

These tests confirm there are three levels of antioxidant activity relating to the extraction solvent used (table 11).

Table 11. Levels of antioxidant potency association with solvent selection. Potency Extraction solvent % inhibition TE mg High Hot water 75-80 36-37 Medium 100% ethanol 56 9-10 25% ethanol Low 45% ethanol 13-33 1-3 60% ethanol

Considering the positive action of the aqueous extracts in the above assays, new extracts were prepared from both dried and fresh leaves (four extractions of each) by hot water decoction as previously described. All samples were adjusted with deionised water to a standard concentration of 10mg/mL. The previous aqueous extract was also compared.

Trolox concentrations produced the curvi-linear response (R2 = 0.98). The fresh leaf decoction was extremely potent (>93% inhibition) followed by the aqueous and water extraction of dried samples (78% inhibition) - Table 12. These results were replicated in a second assay, although the dried leaf decoctions had lower levels of inhibition (62%)

Table 12. Antioxidant activity of hot water decoctions made from fresh (HWG) and dried leaves (HWD). . Solvent TE mg %Inhibition stdev Std err CV AQ 63.10 87.72 0.28 0.11 34.12 HWG 87.10 93.71 0.08 0.03 18.07 HWD 38.02 78.28 0.10 0.05 7.05

22 Summary of all assays conducted on 10mg/ml ethanol extracts The number of assays performed varied between the Ourimbah (OURL-15, OURR-14) and Burnbrae (BBFL-7, BBML-6) specimens according to availability. Mean inhibition rates recorded for 10mg/mL extracts over the period did not differ significantly between specimens, with BBFL the highest (60 %) and OURR the lowest (48%) – see Figure 14, Table 13.

70 Table 13. Antioxidant effects of four D. viscosa specimens. n = number of samples 60 tested 50 % 40 Sample Inhibition SEM n

30 OURL 57.02 3.85 15

20 OURR 48.57 3.35 14 BBML 52.4 6.07 6 10 BBFL 60.25 5.29 7 0 OURL OURR BBML BBFL Figure 14. Mean inhibition ± SEM of AAPH oxidation by D. viscosa samples

4.2.3. Wound healing assay Comparison of proliferation rates in treated and untreated fibroblasts Most samples achieved a high percentage (50-100%) reduction of Alamar Blue compared with controls for the first hour. At the 24hour mark some proliferation was still evident whereas after 48 hours all samples were approaching the same reduction level as the controls. In four of the six samples the lowest dilution (10μg) produced the highest proliferation. Conversely in two samples the highest dilutions (50μg) showed the lowest proliferation rates.

100 90 80 70 60 50 40 30 Relative reduction 20 10 0 010203Time (hours) 04050

10 u g / m l 20 ug/ml 30 ug/ml 40 ug/ml 50 ug/ml

Figure 15. Difference in Alamar Blue reduction between treated and untreated fibroblasts (average of three tests), sample OB507.

23 5. Discussion

The main finding of the regional study is that Burnbrae samples have significantly higher levels of flavonoid-like compounds compared to the other sites. However, the study has built-in limitations. The HPLC method was not calibrated or validated at that time, some sample sizes were small and peak separation and spectrum purity was not well established. These issues were all addressed in subsequent experiments. Taken broadly the results do negate any advantage in sourcing the plant material from more remote areas of the state compared to the more accessible Burnbrae site.

5.1.1 Analysis of chromophores Almost all the peaks submitted to analysis were shown to have similar UV spectra, with maximum absorbances at 335-345nm (Band I) and 270-280nm (Band II). These absorption ranges are typical of 3-OH substituted flavonols, which in turn are the major flavonoid type linked with D. viscosa (Ghisalberti, 1998) (Harborne, 1999). Compound #4 has Band I absorption of 365nm and Band II of 266nm –a pattern typical of non-substituted flavonols at C3 (Markham 1982).

Several known flavonoids were analysed under the same conditions, but none corresponded to the D. viscosa peaks. Further analysis by mass spectrometry and nuclear magnetic resonance would be required in order to identify known compounds or to elucidate the structure of any previously unknown compounds that may be present.

5.1.2 Quercetin calibration Without confirmation of specific flavonoid-like compounds in the D. viscosa samples, quantification of flavonoids was achieved by adopting quercetin as a calibration standard. In the initial application of this method QE values were determined for a wide variety sample types. Flavonoid levels from aqueous extracts were consistently low, hence ethanol extracts were selected for conducting comparisons between sites and in different months. Initial tests indicated considerable variability in total flavonoid levels between and within sites, most particularly between Ourimbah L and R where the big QE difference can be traced to two peaks (#5 and #10) – see Table 6. The fact that flavonoid levels in all flower and capsule samples were very low supports the continued use of leaves as the basis for therapeutic applications - particularly given their year round availability and abundance.

Flavonoid levels from leaves were compared with Getie’s data, of 2.65μg/mg, 1.59μg/mg and 15.33 μg/mg for quercetin, kaempferol and isorhamnetin respectively (Getie et al., 2000). These are in a similar order of magnitude to the current findings, although direct comparisons cannot be made with specific compounds. However it does indicate that D. viscosa 100% ethanol extracts contain sufficient levels of flavonoids to activate wound-healing mechanisms.

5.1.3 Environmental factors and flavonoid variability It is necessary to understand the relationship between environmental factors and phytochemical production in order to optimize field growth conditions (Briskin, 2000) and selection of breeding stock for medicinal plants (Vanhaelen et al., 1991). Flavonoid variability in plants has been a specific focus of research due to the unique role these flavonoids play in moderating environmental factors in general and UV-B exposure in particular. Some species show a strong bias towards accumulation of flavonoids in certain months or seasons, indicating the influence of climatic factors as well as UV-B exposure (Chaves and Escudero, 1999). The results of this study give little indication of seasonal changes, although QE levels during the winter have yet to be tested. Other factors do appear to correlate with variable flavonoid levels, including extraction solvent and plant section tested.

Individual specimen selection also appears to correlate with quercetin levels particularly at the Ourimbah site. However the results do not necessarily indicate that harvesting site is a factor, given the variation between the two Ourimbah specimens is greater than any variation between specimens at the two sites. The study also demonstrates that young seedlings may be transplanted from their wild habitat at Burnbrae to a distant site (Ourimbah) with different weather conditions, without any reduction in flavonoid levels.

24

5.2 Anti-inflammatory activity No inhibition of Cox-2 activity was demonstrated by any extracts at the concentrations tested, suggesting that D.viscosa does not work at this part of the inflammatory pathway. A further test to investigate the influence on PGE2 production also showed there was no activity. However there are possible alternative mechanisms of anti-inflammatory activity, such as the alternate arachidonic acid metabolite – 5-lipoxygenase (Bauer, 1999). Studies are currently underway to further investigate this possibility.

5.3 Antioxidant assays Many degenerative diseases, including those of an inflammatory nature, are linked to oxidative stress. Oxidative stress results from cumulative damage caused by reactive oxygen species (ROS) including free radicals (with unpaired electrons) or their non-radical derivatives such as hydrogen peroxide and singlet oxygen (Hughes, 2002). Demonstration of free radical inhibition could offer an alternative mechanism for moderating inflammatory processes.

5.3.1 Spectrophotometer assay All extracts tested provided some antioxidant effects when compare to the vitamin E analogue Trolox. The hot water and higher strength ethanol extracts showed the greatest activity, approaching 100% inhibition of peroxyl radicals. In terms of concentration, the ethanol extracts show a strong relationship (means inhibition 18% at 5mg/mL - 95% at 40mg/mL) while cold water extracts show an inverse relationship - the highest concentration gives the least antioxidant effect and visa versa. However the antioxidant potency of cold water extracts is relatively weak in this assay.

When comparing the means of all extracts by sample type (ethanol, hot water, cold water and Trolox) using one way ANOVA, there are significant differences between them: F(3, 121) = 50.4 p<.0001.

5.3.2 Microplate assay While there was little variation between specimens and sites, considerable variation was found when different extraction solvents were used. As with the spectrophotometric method, the greatest antioxidant activity was provided by hot water extracts followed by 100% and 25% ethanol extracts, while those prepared with 45% and 60% ethanol provided the weakest activity.

The reason for this phenomenon is not clear. The high flavonoid levels found in the 100% ethanol extracts provide a rationale for their activity, however the aqueous and 25% ethanol extracts are relatively low in flavonoids. Hence there must be water-soluble constituents involved – possibly saponins. The 45% and 60% ethanol extracts may contain insufficient levels of either the flavonoids or the water soluble constituents to assert the same level of antioxidant activity as the other extracts, although their activities are still of a comparable potency to Trolox.

5.4 Wound healing assay D. viscosa preparations have been widely used in Australia for wound healing. It is possible this activity is secondary to other actions such as anti-inflammatory, antioxidant and antimicrobial. Documented analgesic and antipruritic properties could also enhance the efficacy as a general healer of wounds and skin disorders in general. Inflammation is but one transient phase of three overlapping phases associated with wound healing, the others being tissue formation and tissue remodelling (Singer and Clark, 1999). In order to assess the direct effects of D. viscosa on the skin, a fibroblast proliferation model to assess potential wound-healing activity was conducted.

Initial results suggest D. viscosa extracts (100% and 45% ethanol) have a strong proliferative effect on fibroblast growth as determined by the degree of Alamar Blue reduction. However a concentration response has not been observed, although the initial results suggest the lower concentrations are more potent. The control assay conducted in the absence of fibroblasts refutes the possibility that Alamar Blue reduction may be induced by antioxidant components of the test extracts or by mitochondrial enzymes.

25 6. Conclusions and Recommendations

D. viscosa leaf extracts are a rich source of flavonoids and flavonoid-like compounds, although their individual levels as measured by quercetin equivalents may vary between individual specimens. Leaves from one site (Burnbrae) consistently provided high flavonoid levels compared to other sites tested in the regional survey. However, one seedling transplanted to the Ourimbah campus from Burnbrae produced higher levels of QEs after only two years growth.

o Ongoing monitoring for seasonal variation in flavonoid levels at the two sites is required to gain a longer-term perspective and minimise possible influences of weather fluctuations from year to year.

o Further phytochemical analysis is required to assess presence of non-flavonoid compounds - especially saponins and diterpenes.

Ethanol extracts were shown to contain levels of flavonoids of the magnitude thought necessary to promote wound healing and inhibit free radical activity. While D. viscosa appears to have no influence on Cox-2 or PGE2 activity, it has demonstrated other properties that may form the basis of anti- inflammatory activity.

o The lipoxygenase assay using a linoleic acid substrate should be completed in a further attempt to demonstrate a mechanism of anti-inflammatory activity.

All D. viscosa extracts are able to reduce peroxidation of AAPH in vitro, however, hot water extracts have the most potent antioxidant capacity in both the spectrophotometer and microplate methods, despite containing low flavonoid levels. Initial studies reveal that extracts prepared with ethanol (100% and 45%) are able to stimulate fibroblast growth when compared to controls, however aqueous extracts are yet to be tested by this method.

o Further testing of D. viscosa extracts (including aqueous extracts) in the fibroblast proliferation assay is required, to further substantiate potential wound healing properties, and to establish a concentration – response range.

Good quality plant material from D. viscosa var. angustifolia is relatively abundant, and is easy to harvest and process. It appears to thrive under cultivation, and the phytochemical matrix and biological activities do not appear to be unduly influenced by transplanting or relocating. The species has very desirable characteristics for exploitation by primary producers.

o Small field and greenhouse trials are required to optimise growth conditions and build up stocks in anticipation of future demand. Specimens should be continually monitored for flavonoid levels, as measured by QEs.

o Formulation of topical products based on both aqueous and ethanol leaf extracts is required for small clinical tests on humans.

o The findings of this project could be used as part of an application to have the species considered for listing on the Australian Register of Therapeutic Goods. This will require further financial commitment and approximately 12–24 months to acquire toxicology data, paving the way for further work in product formulation.

26 References

Abdel-Mogis, M. B., S.A. Asiri, A.M. Sobahi, T.R. and Batterjee, S.M. 2001 New clerodane diterpenoid and flavonol-3-methyl ethers from Dodonaea viscosa Pharmazie, 56, 830-831. Bauer, R. 1999 In Bioassay Methods in Natural product Research and Drug development, Vol. 43 (Ed, Bohlin, L. B., J.G.) Kluwer Academic Publishers, Dordrecht, pp. 119-141. Bioscource 2005. Alamar Blue Assay. Company Literature, USA. Briskin, D. P. 2000 Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health Plant Physiology, 124, 507-514. Brolis, M. G., B. et al. 1998 Identification of HPLC-diode array-mass spectrometry and quantification by HPLC-UV absorbance detection of active constituents of Hypericum perforatum J Chromatography, 825, 9-16. Butler, M. S. 2004 The Role of Natural Product Chemistry in Drug Discovery J. Nat. Prod., 67, 2141- 2153. Chaves, N. E., J.C. 1999 In Principles and practices in plant ecology(Ed, Inderjit, K., K.M.M. & Foy, C.L.) CRC Press, Boca raton. Cooper, J. G., C 1972 Tutorial Pharmacy, Pitman Medical, London. Dunlap, W. L., L. Doyle, J. & Yamamoto, Y. 2003 A microtiter plate assay for screening antioxidant activity in extracts of marine organisms. Marine Biotechnology, 5, 294-301. El-Zwi, M. A. a. A., A.F.S. 1999 Chemical studies on the contents of Dodonaea viscosa (flowers) and Agaricus sp. Chemical Environmental Research, 8, 285-288. Getie, M. G.-M., T. Rietz, R. & Neubert, R.H.H 2000 Distribution of quercetin, kaempferol and isorhamnetin in some Ethiopian medicinal plants used for the treatment of dermatological disorders. Ethiopian Pharmacy Journal, 18, 25-34. Ghisalberti, E. L. 1998 Ethnopharmacology and phytochemistry of Dodonaea species Fitoterapia, LXIX, 99-113. Graz, B., Elisabetsky, E. and Falquet, J. 2007 Beyond the myth of expensive clinical study: Assessment of traditional medicines Journal of Ethnopharmacology, 113, 382-386. Harborne, J. B. e. 1999 The handbook of natural flavonoids, John Wiley & Sons, Chichester. Hughes, D. A. 2002 In Nutrition and Immune Function(Ed, Calder, P. C. F., C.J. & Gill, H.S.) CABI, Wallingford. Khalil, N. M., Sperotto, J. S. and Manfron, M. P. 2006 Antiinflammatory activity and acute toxicity of Dodonaea viscosa Fitoterapia, 77, 478-480. Khan, M. S., Kalim Javed & Hasnain Khan, M 1992 Constituents of the flowers of Dodonea viscosa Fitoterapia, LXIII, 83-84. Krishnan, P. 2006 The scientific study of herbal wound healing therapies: Current state of play Current Anaesthesia & Critical Care, 17, 21-27. Kumar, B., Vijayakumar, M., Govindarajan, R. and Pushpangadan, P. 2007 Ethnopharmacological approaches to wound healing--Exploring medicinal plants of India Journal of Ethnopharmacology, 114, 103-113. Markham, K. R. 1982 In Techniques of Flavonoid AnalysisAcademic Press, London, pp. 36-51. Mata, R. C., J.L. Cristanto, D. Pereda-Miranda, R. and Castaneda, P. 1991 New secondary metabolites from Dodonaea viscosa J Nat Prod, 54, 913-917. Miller, J. M. 2005 In Chromatography: Concepts and contrastsJohn Wiley & Sons. Naranjo, P. 1995 In Ethnobotany, evolution of a discipline(Ed, Reis, R. E. S. a. S. v.) Timber press, Portland, pp. 363. O'Brien, J., Wilson, I., Orton, T. and Pognan, F. 2000 Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity European Journal of Biochemistry, 267, 5421-5426. Painter, G. 1998 A herbalists medicine-making workbook, Gilian Painter, Auckland. Pearman, G. 2000 In AnthropologyUniversity of Kent, Canterbury, pp. 40. Pengelly, A. 2003 In NHAA 5th International Conference on PhytotherapeuticsNational Herbalists Association of Australia, Canberra, pp. 20.1 - 20.9.

27 Sachdev, K. and Kulshreshtha, D. K. 1983 Flavonoids from Dodonaea viscosa Phytochemistry, 22, 1253-1256. Sachdev, K. and Kulshreshtha, D. K. 1986 Viscosol, a c-3' prenylated flavonoid from Dodonaea viscosa Phytochemistry, 25, 1967-1969. Siddiqui, A. A. 1998 Chemical and pharmacological evaluation of Dodonaea viscosa Asian Journal of Chemistry, 10, 14-16. Singer, A. J. and Clark, R. A. F. 1999 Cutaneous Wound Healing N Engl J Med, 341, 738-746. Slikkerveer, L. J. 2006 In Medicinal and Aromatic Plants, Vol. 17 (Ed, Bogers, R. J. C., & Lange, D.) Springer, Dordrecht, pp. 1-28. Vanhaelen, M. L., J. Hanocq, M & Molle, L. 1991 In The medicinal Plant Industry.(Ed, Wijesera., R.) Florida, pp. CRC Press. Wagner, H. 2005 In Handbook of Medicinal Plants(Ed, Yaniv, Z. B., U.) Haworth press, New York. Warren, R. 1990 Australian plants are our neglected resource Australian Horticulture, 28-32. Whittle, B. J. R. 2004 Mechanisms underlying intestinal injury induced by anti-inflammatory COX inhibitors European Journal of Pharmacology, 500, 427-439. Wollenweber, E. 1993 In The Flavonoids: Advances in research since 1986.(Ed, J.B.Harborne) Chapman & Hall, London. Wollenweber, E. and Roitman, J. N. 2007 New reports on surface flavonoids from Chamaebatiaria (Rosaceae), Dodonaea (), Elscholtzia (Lamiaceae) and Silphium (Asteraceae) Natural products Communications, 2, 385-389.

28 Appendix 1. Flavonoids of Dodonaea viscosa

Common name Chemical name Structure UV spectra Reference Gisalberti No #

OMe -

HO O γ ,

OM e OMe OH O

Viscosol - a prenylated trimethoxyflavone 337, 300sh, 270 Viscosol 3’-( γ dimethyal llyl)-5,7- dihydroxy -3,6,4’- trimethox yflavone and (Sachdev Kulshreshtha, 1986) 5,7- dihydroxy -3’- (3- hydroxym ethylbutyl )-3,6,4’- trimethox yflavone 338, 275 Sachdev & Kulshreshtha 1983 60

OCH3

H CO 3 O

H3CO OCH3

OH O 5- hydroxy- 3,6,7,4’- tetrameth oxyflavon e 58

OH

HO O

H3CO OCH3

OH O 5,7,4’- trihydrox y-3,6- dimethox yflavone 340, 273 Sachdev & Kulshreshtha 1983 Van Heerden et al.,2000 59

HO O

OH O pinocembri n 5,7- dihydroxyfl avanone Sachdev & Kulshreshtha 1983

3

HO O

H3CO OCH3

OH O Santin 5,7- dihydroxy - 3,6,4’3’ tetramethox yflavone (trimethoxy flavone) 340, 274 Sachdev & Kulshreshtha 1983 & Harborne 1999 Baxter Aliarin 5,7,4’- trihydroxy- 3’- (3 hydroxymet hylbutanol) 3,6- dimethoxyfl avone 335, 300sh, 272 Sachdev & Kulshreshtha 1983 62 5,7- dihydroxy- 3’- (3 hydroxymet hylbutanol) 3,6,4’- trimethoxyfl avone Sachdev & Kulshreshtha 1983 63 5,4’- dihydroxy- 3’- (3 hydroxymet hylbutanol) 3,5,6,4’- tetramethox yflavone 350 305sh 270 Sachdev & Kulshreshtha 1983 64

29 Isokaempfer Ermanin Kumatakeni Rhamnocitri Genkwanin Cirsimaritin Pectolinarig penduletin ide (Kaempfero n n (Apigenin (Scutellarei enin l 3,4’ – (Kaempfero (Kaempfero 7- n 6,7- (Scutellarei dimethyl l 3,7 – l 7– methyl methoxyfla dimethyoxy n 6,4’- ether) dimethyl ether vone) flavone) dimethyoxy ether) flavone)

3, 5,6,7, 4’- 3,5,7,4’- 3,5,7,4’- 3,5,7,4’- 3,5,7,4’- 3,5,7,4’- 3,5,7,4’- 5,7,4’- 5, 4’- 5, 7 - 5,4’– 3’- (3 Pentahydro tetrahydrox Tetrahydrox Tetrahydrox Tetrahydrox Tetrahydrox Tetrahydrox Trihydroxy- Dihydroxy- Dihydroxy- Dihydroxy- hydroxymet xyl 3,6,7,4’- yl-3,7,4’- yl-7,4’- yl-3’- yl-3,4’- y-3,7- y-7- 7- 6,7- 6, 4’- 3,6,7- hylbutanol) tetramethox trimethoxyfl dimethoxyfl methoxyfla dimethoxyfl dimethoxyfl methoxyfla methoxyfla dimethoxyfl dimethoxyfl trimethoxyfl 3,5,6,7,4’- yflavone avone avone vone avone avone vone vone avone avone avone pentametho xyflavone H H H H H H HO HCO H 3 3 3 3 3 3 CO CO CO 3 CO HO CO CO CO OH OH OH OH OH OH OH O 30 O O O O O O O O

O O O O OCH OH OCH 3 OCH 3 3 OH O O O O C H O H C H H H 3

3

340, 272

(Wollenwebe (Wollenwebe (Wollenwebe (Wollenwebe (Wollenwebe (Wollenwebe (Wollenwebe Wollenweber (Wollenwebe (Wollenwebe Sachdev & Sachdev & r, 1993) r, 1993) r, 1993) r, 1993) r, 1993) r, 1993) r, 1993) & Roitman r, 1993) r, 1993) Kulshreshtha Kulshreshtha 2007 1983 1983

70 57 65 6 Kaempferol Acacetin7- Sakuranetin Isorhamneti Isorhamneti Isorhamneti Kaempferol Kaempferol Quercetin Hydroxykae 3 – methyl methyl ether n 3- n 3- n 3- 7,4’- 3,7,4’- mpferol – ether rhamnosylg rutinoside rhamnoside dimethyl trimethyl trimethyl alactoside ether ether ether (penduletin? ) 6 Hydroxy 5,7,4’ 5 hydroxy - (S)-5,4’- 5,6,4’- 5,7,3’,4’,- 5,7,3’,4’,- 5,7,4’,- 3,5- 5-Hydroxy- 3,5,7,3’,4’- – 3,6,7 - Trihydroxy 7,4’- dihydroxy- triydroxy- tetrahydrox Tetrahydrox Trihydroxy- Dihydroxy- 3,7,4’- Pentahydro trimethoxyfl -3- dimethoxyfl 7- 3,7,- yl-3- yl-3- 3-rutinoside 7,4’- trimethoxyfl xyflavone avone methoxyfla avone methoxyfla dimethoxyfl rutinoside galactoside dimethoxyfl avone vone vone avone ?? avone HO H H H 3 3 3 CO CO CO OH OH OH OH 31 O O O O

O O O O OCH OCH OH 3 3 O C O O H C C 3 H H 3 3

Wollenweber Wollenweber (Abdel-Mogis (Mata et al., (Sachdev and (Harborne, Harborne & & Roitman & Roitman et al. , 2001) 1991) Kulshreshtha, 1999) Baxter 1999 2007 2007 1983)

71 69 68 67 72 56 66

Narigenin Narigenin 7 Eriodictyol Eriodictyol 7,4’ – – methyl 7,3’ – 7 – methyl dimethyl ether dimethyl ether ether ether

5,7,4 - 5,7,4 - 5,7,3’,4’ 5,7,3’,4’ trihydroxy – trihydroxy – Tetrahydrox Tetrahydrox 7,4’ - 7 - y -7,3’- y -7- dimethyoxy methyoxyfl dimethoxyfl methoxyfla flavanone avanone avone vone H H H H 3 3 3 3 CO CO CO CO OH OH OH OH 32 O O O O O O O O

OCH O O O H H H 3

(Wollenwebe Wollenweber Wollenweber Wollenweber r and & Roitman & Roitman & Roitman Roitman, 2007 2007 2007 2007)

Medicinal Activity of Dodonaea viscosa —A preliminary study—

RIRDC Publication No. INSERT PUB NO. HERE RIRDC Publication No. 08/172

This study assesses the potential anti-inflammatory properties The Rural Industries Research and Development Corporation of extracts of Dodonaea viscosa, an indigenous plant species (RIRDC) manages and funds priority research and translates previously used in traditional medicine, as a topical anti- results into practical outcomes for industry. inflammatory product. Our business is about new products and services and better ways The publication provides information to enable the of producing them. Most of the information we produce can be manufacture of a product that utilises locally grown raw downloaded for free from our website: www.rirdc.gov.au. material and encourages alternative crops of D. viscosa within the primary producing sector so that an innovative product for RIRDC books can be purchased by phoning 02 6271 4100 or the complementary medicine sector can be produced. online at: www.rirdc.gov.au/eshop.

This research confirms that Australian D. viscosa has sufficient levels of active constituents to account for its reputation as a traditional medicine. It is readily harvested and extracts prepared using simple techniques have demonstrated antioxidant and wound healing activity in vitro.

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