Andrew Pengelly



Demonstration of in vitro antioxidant and wound healing activities in Australian Dodonaea viscosa Jacq. subsp. angustifoliaAndrew Pengelly, 2011AbstractThe Australian medicinal plant Dodonaea viscosa subsp. Angustifolia was subjected to antioxidant and in vitro biological assays, to assess therapeutic potential for localised inflammatory disorders and to assist wound healing. To this effect, antioxidant activity was tested using an in vitro peroxidation kinetics assay, anti-inflammatory activity was tested using a lipoxygenase (LOX) assay, and wound healing potential was assessed by cytotoxicity and cultured fibroblast proliferation assays. D. viscosa extracts exerted potent antioxidant activity whether measured as percentage of oxidation inhibition compared to control or as Trolox equivalents (TE). The 100% ethanol. extract demonstrated a linear dose-dependent inhibition on LOX activity, but the aqueous and aqueous-ethanol extracts were devoid of activity. However, both aqueous and ethanolic extracts demonstrated proliferative effect on cultured human dermal fibroblasts. Methanol and acetone extracts demonstrated cytotoxic effects towards cultured mouse lymphoblasts and human melanoma cells at high doses, while aqueous extracts showed minimal cytotoxicity. The findings of this study collectively indicate the species has substantial antioxidant and wound healing activity, and consideration should be given towards initiating studies on humans.IntroductionIn response to public demand for natural alternatives to conventional wound healing treatments and for products that relieve pain and inflammation, clinical and experimental research into the biological activity of traditional plant medicines is occurring around the world, via the emerging discipline of ethnopharmacology. In the past, indigenous Australians were exposed to attack from snakes, insects, jellyfish and stonefish as well as burns from campfires and other general accidents, and Dodonaea viscosa is one of the best documented plants for treatment of injuries of this kind in Australia ADDIN EN.CITE <EndNote><Cite><Author>Cribb</Author><Year>1981</Year><RecNum>178</RecNum><MDL><REFERENCE_TYPE>1</REFERENCE_TYPE><REFNUM>178</REFNUM><AUTHORS><AUTHOR>Cribb, A.B. </AUTHOR><AUTHOR>Cribb, J.W.</AUTHOR></AUTHORS><YEAR>1981</YEAR><TITLE>Wild Medicine in Australia</TITLE><PLACE_PUBLISHED>Sydney</PLACE_PUBLISHED><PUBLISHER>Collins</PUBLISHER><LABEL>Hopbush general</LABEL></MDL></Cite><Cite><Author>Aboriginal</Author><Year>1993</Year><RecNum>176</RecNum><MDL><REFERENCE_TYPE>1</REFERENCE_TYPE><REFNUM>176</REFNUM><AUTHORS><AUTHOR>Aboriginal, </AUTHOR><AUTHOR>Community,N.T.</AUTHOR><AUTHOR>Australia</AUTHOR></AUTHORS><YEAR>1993</YEAR><TITLE>Traditional Aboriginal Medicines</TITLE><PLACE_PUBLISHED>Darwin</PLACE_PUBLISHED><PUBLISHER>Conservation Commission of the Northern territory</PUBLISHER><LABEL>Hopbush general</LABEL></MDL></Cite></EndNote>(Cribb and Cribb 1981; Aboriginal 1993). D. viscosa is polymorphic and is found across much of the Australian continent, with individual subspecies having marked regional distribution patterns. Beyond Australia D. viscosa is found as far afield as Central and South America, Florida, Ethiopa, South Africa, India, Taiwan, as well as New Zealand and numerous Pacific islands. The species is unique in terms of its widespread geographical distribution over six continents, and associated variations in patterns of human use around the world have been observed (Pearman 2000). The subspecies angustifolia is relatively abundant in the Hunter Valley region of Australia, and extracts and creams made from it have provided preliminary clinical evidence of significant anti-inflammatory and analgesic activity (Pengelly 2003) ADDIN EN.CITE <EndNote><Cite><Author>Pengelly</Author><Year>2003</Year><RecNum>196</RecNum><MDL><REFERENCE_TYPE>3</REFERENCE_TYPE><REFNUM>196</REFNUM><AUTHORS><AUTHOR>Andrew Pengelly</AUTHOR></AUTHORS><YEAR>2003</YEAR><TITLE>The phytochemistry and bioactivity of Dodonaea viscosa (hopbush)</TITLE><SECONDARY_TITLE>NHAA 5th International Conference on Phytotherapeutics</SECONDARY_TITLE><PLACE_PUBLISHED>Canberra</PLACE_PUBLISHED><PUBLISHER>National Herbalists Association of Australia</PUBLISHER><PAGES>20.1 - 20.9</PAGES><LABEL>Hopbush general</LABEL></MDL></Cite></EndNote>. In his review of the Dodonaea genus, Gisalberti (1998) notes similarities in reported medicinal uses by indigenous people in different parts of the world. The well-recorded traditional uses suggest it is safe – there are no recordings of adverse reactions. Studies on animals confirm a high level of safety with no acute toxicological signs manifested for D. viscosa preparations ADDIN EN.CITE <EndNote><Cite><Author>Khalil</Author><Year>2006</Year><RecNum>141</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>141</REFNUM><YEAR>2006</YEAR><TITLE>Antiinflammatory activity and acute toxicity of Dodonaea viscosa</TITLE><SECONDARY_TITLE>Fitoterapia</SECONDARY_TITLE><VOLUME>77</VOLUME><NUMBER>6</NUMBER><PAGES>478-480</PAGES><DATE>2006/9</DATE><AUTHORS><AUTHOR>Khalil, N.M.</AUTHOR><AUTHOR>Sperotto, J.S.</AUTHOR><AUTHOR>Manfron, M.P.</AUTHOR></AUTHORS><URL> viscosa</KEYWORD><KEYWORD>Antiinflammatory activity</KEYWORD><KEYWORD>Acute toxicity</KEYWORD></KEYWORDS><LABEL><styles></styles>Anti-inflammatory models</LABEL></MDL></Cite><Cite><Author>Arun</Author><Year>2008</Year><RecNum>314</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>314</REFNUM><AUTHORS><AUTHOR>M. Arun</AUTHOR><AUTHOR>V.V. Asha</AUTHOR></AUTHORS><YEAR>2008</YEAR><TITLE>Gastroprotective effect of Dodonaea viscosa on various experimental ulcer models</TITLE><SECONDARY_TITLE>J Ethnopharmacology</SECONDARY_TITLE><VOLUME>118</VOLUME><PAGES>460-465</PAGES></MDL></Cite></EndNote>(Khalil et al. 2006; Arun and Asha 2008; Teshome et al 2010) ADDIN EN.CITE <EndNote><Cite><Author>Khalil</Author><Year>2006</Year><RecNum>141</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>141</REFNUM><YEAR>2006</YEAR><TITLE>Antiinflammatory activity and acute toxicity of Dodonaea viscosa</TITLE><SECONDARY_TITLE>Fitoterapia</SECONDARY_TITLE><VOLUME>77</VOLUME><NUMBER>6</NUMBER><PAGES>478-480</PAGES><DATE>2006/9</DATE><AUTHORS><AUTHOR>Khalil, N.M.</AUTHOR><AUTHOR>Sperotto, J.S.</AUTHOR><AUTHOR>Manfron, M.P.</AUTHOR></AUTHORS><URL> viscosa</KEYWORD><KEYWORD>Antiinflammatory activity</KEYWORD><KEYWORD>Acute toxicity</KEYWORD></KEYWORDS><LABEL><styles></styles>Anti-inflammatory models</LABEL></MDL></Cite><Cite><Author>Arun</Author><Year>2008</Year><RecNum>314</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>314</REFNUM><AUTHORS><AUTHOR>M. Arun</AUTHOR><AUTHOR>V.V. Asha</AUTHOR></AUTHORS><YEAR>2008</YEAR><TITLE>Gastroprotective effect of Dodonaea viscosa on various experimental ulcer models</TITLE><SECONDARY_TITLE>J Ethnopharmacology</SECONDARY_TITLE><VOLUME>118</VOLUME><PAGES>460-465</PAGES></MDL></Cite></EndNote>, however to date there have been no clinical trials to formally assert claims of safety and efficacy on humans.D.viscosa contains di- and triterpenes, saponins, flavonoids, coumarins and other phenolic compounds. It is likely that 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). However phytochemical investigations suggest that three specific compound groups – diterpenes, saponins and flavonoids – are responsible for the majority of biological activity associated with the species (Ghisalberti 1998; ADDIN EN.CITE <EndNote><Cite><Author>Siddiqui</Author><Year>1998</Year><RecNum>77</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>77</REFNUM><AUTHORS><AUTHOR>Siddiqui, A.A.</AUTHOR></AUTHORS><YEAR>1998</YEAR><TITLE>Chemical and pharmacological evaluation of Dodonaea viscosa</TITLE><SECONDARY_TITLE>Asian Journal of Chemistry</SECONDARY_TITLE><VOLUME>10</VOLUME><NUMBER>1</NUMBER><PAGES>14-16</PAGES><LABEL><styles></styles>Hopbush chemistry</LABEL></MDL></Cite></EndNote>Siddiqui 1998).Diterpenes include hautriwaic acid, dodonic acid, several ent-labdane diterpenes and a recently discovered group known as methyl dodonates A, B and C (Ortega et al. 2001). Over 30 flavonoids have been identified, including methoxyflavones such as 5-hydroxy-3,6,7,4’-tetramethoxyflavone, pinocembrin, santin, alizarin and penduletin as well as widely distributed compounds such as quercetin and isorhamnetin ADDIN EN.CITE <EndNote><Cite><Author>Sachdev</Author><Year>1983</Year><RecNum>81</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>81</REFNUM><AUTHORS><AUTHOR>Sachdev, K.</AUTHOR><AUTHOR>Kulshreshtha, D.K.</AUTHOR></AUTHORS><YEAR>1983</YEAR><TITLE>Flavonoids from Dodonaea viscosa</TITLE><SECONDARY_TITLE>Phytochemistry</SECONDARY_TITLE><VOLUME>22</VOLUME><NUMBER>5</NUMBER><PAGES>1253-1256</PAGES><LABEL>Hopbush chemistry</LABEL></MDL></Cite></EndNote>(Sachdev and Kulshreshtha 1983; Wollenweber 1993; Teffo, Aderogba and Eloff 2010).A B Figure 1. Constituents of D. viscosa. A. Methyl dodonate A - a tricyclic clerodane diterpene B. Penduletin, a typical 3-OH substituted flavonoid. D. viscosa contains polyhydroxylated triterpenes based on oleanolic acid, including jegosapogenol and barrigenol and their esters (Dimbi et al. 1985). These structures also form glycosides, known as dodonosides, which provide typical saponin activity (detergent, antimicrobial and anti-inflammatory) (Wagner et al. 1987). More recently specimens from Madagascar yielded two new oleanane-type triterpenoid saponins with antiproliferative activity against human ovarian cancer cells (Cao et al 2009).D. viscosa is used in Ethiopia for treating inflammatory skin disorders. This activity has been linked to the presence of relatively large concentrations of three flavonoids - quercetin, kampferol and isorhamnetin (Getie et al. 2000). These investigators performed an in vitro multilayer membrane test to demonstrate that these flavonoids are effectively released from a hydrophilic cream base. However, there was practically no release from a lipophilic cream. Despite the fact D. viscosa has been used as a traditional wound healing remedy in many countries including Ethiopia, Mexico, India and Australia, wound healing activity has not been scientifically validated for the species apart from Getie’s work in Ethiopia ADDIN EN.CITE <EndNote><Cite><Author>Getie</Author><Year>2002</Year><RecNum>96</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>96</REFNUM><AUTHORS><AUTHOR>Getie, M. Gebre-Mariam, T. Rietz, R. &amp; Neubert, R.H.H</AUTHOR></AUTHORS><YEAR>2002</YEAR><TITLE>Evaluation of the release profiles of flavonoids from topical formulations of the crude extract of the leaves of Dodonaea viscosa (Sapindaceae).</TITLE><SECONDARY_TITLE>Pharmazie</SECONDARY_TITLE><VOLUME>57</VOLUME><PAGES>320-322</PAGES><LABEL><styles></styles>Hopbush chemistry</LABEL></MDL></Cite></EndNote>(Getie et al. 2002). Since regeneration of healthy tissues adjacent to inflamed lesions and wounds is dependent upon the proliferation of fibroblasts ADDIN EN.CITE <EndNote><Cite><Author>Mosley</Author><Year>2004</Year><RecNum>281</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>281</REFNUM><AUTHORS><AUTHOR>Mosley, R.</AUTHOR><AUTHOR>Hilton, J.R. </AUTHOR><AUTHOR>Waddington, R.J.</AUTHOR><AUTHOR>Harding, K.G.</AUTHOR><AUTHOR>Stephens, P. </AUTHOR><AUTHOR>Thomas, D.W.</AUTHOR></AUTHORS><YEAR>2004</YEAR><TITLE>Comparison of oxidative stress biomarker profiles between acute and chronic wound environemtns</TITLE><SECONDARY_TITLE>Wound Repair and Regeneration</SECONDARY_TITLE><VOLUME>12</VOLUME><NUMBER>4</NUMBER><PAGES>419-429</PAGES><LABEL>Wound &amp; injury healing</LABEL></MDL></Cite></EndNote>(Mosley et al. 2004), a fibroblast cell culture model is often used to assess wound healing potential. In this system the ability of substances or extracts to induce the proliferation of human dermal fibroblasts in culture is determined. The current study was conducted to assess whether Dodonaea viscosa subsp. angustifolia could provide the biological activity required for combating localised inflammatory disorders such as dermatitis by the application of antioxidant, lipoxygenase (LOX) and cytotoxic assays, and whether it could additionally provide fibroblast proliferation activity. MethodsPlantsDodonaea viscosa subsp. angustifolia specimens were harvested from the Upper Hunter Valley, New South Wales. Voucher specimens were authenticated by Dr. Judy West and deposited at the Australian National Herbarium. Harvested leaves were dried in a dehydrator (BBO Forced air system, G.T.D. Pty. Ltd., Sydney) at 38.5°C, and ground using a mortar and pestle. Samples were extracted in either 100% absolute ethanol or various aqueous-ethanol solutions as required at a concentration of 1% w/v. All extracts were placed in a Netrasonik 57X water bath sonicator for 5 minutes.Antioxidant activityAntioxidant activity was tested using an oxidative kinetics model, in which samples of leaf extracts were subjected to a peroxidation assay. Oxidation inhibition was assessed using percentage inhibition of control or as Trolox equivalents (TE), based on the water-soluble vitamin E analogue Trolox. 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).Trizma-hydrochloride (Tris-HCl), ethylenediaminetetra-acetic acid (EDTA) - ACS reagent grade, dihydrorhodamine-123 (DHR), sodium hydroxide and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylix acid) were purchased from Sigma Chemical Co. , St. Louis, MO USA. (2,2’- azobis(2-aminopropane) dihydrochloride (AAPH) was purchased from Wako Pure Chemical Industries (Osaka, Japan).The assay described for use with a spectrophotometer can also be conducted on a 96-well microplate reader once the sample and reagent volumes are scaled down according to Dunlap’s alternative assay, to achieve reagent concentrations of 50μM DHR and 2 mM AAPH in each microplate well ADDIN EN.CITE <EndNote><Cite><Author>Dunlap</Author><Year>2003</Year><RecNum>52</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>52</REFNUM><AUTHORS><AUTHOR>Dunlap, W.,</AUTHOR><AUTHOR>Llewellyn, L.,</AUTHOR><AUTHOR>Doyle, J.,</AUTHOR><AUTHOR>Yamamoto, Y.</AUTHOR></AUTHORS><YEAR>2003</YEAR><TITLE>A microtiter plate assay for screening antioxidant activity in extracts of marine organisms.</TITLE><SECONDARY_TITLE>Marine Biotechnology</SECONDARY_TITLE><VOLUME>5</VOLUME><PAGES>294-301</PAGES><LABEL>Antioxidant models</LABEL></MDL></Cite></EndNote>(Dunlap et al. 2003). Trolox dilutions commonly employed were 0, 37.5, 75, 150 and 300uM. Absorbance was read on a microplate reader (Benchmark +, Bio-Rad, Sydney). Lipoxygenase inhibition assayPotassium tetraborate, tween 20, linoleic acid and soy lipoxidase lyophilised powder (80,000 units/mg) were all purchased from Sigma, St. Louis, MO, USA.To test for anti-inflammatory properties, D. viscosa extracts were tested for effects on lipoxygenase (LOX), an important enzyme in the inflammatory pathway. This procedure was based on the soybean 5-LOX assay developed by Sircar et al. (1983) as modified by Evans ADDIN EN.CITE <EndNote><Cite><Author>Evans</Author><Year>1991</Year><RecNum>181</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>181</REFNUM><YEAR>1991</YEAR><TITLE>Natural products as probes for new drug target identification</TITLE><SECONDARY_TITLE>Journal of Ethnopharmacology</SECONDARY_TITLE><VOLUME>32</VOLUME><NUMBER>1-3</NUMBER><PAGES>91-101</PAGES><DATE>1991/4</DATE><AUTHORS><AUTHOR>Evans, Fred J.</AUTHOR></AUTHORS><URL> general</LABEL></MDL></Cite></EndNote>(1991). The method was conducted with LOX derived from soybean meal, its activity was monitored on a Cary 50 spectrophotometer at 234 nm, using linoleic acid as the substrate. Different concentrations of ethanolic and aqueous D. viscosa samples (150μL) were added to reagents in cuvettes. Blank controls (150μL) consisted of the reagents plus equivalent volumes of extraction solvents. The enzyme reaction was commenced by adding enzyme to the linoleic acid substrate. Using a spectrophotometer, the increase in absorption at 234nm due to formation of lipid peroxide (conjugated diene) from the linoleic acid was recorded over 30 minutes ADDIN EN.CITE <EndNote><Cite><Author>Young</Author><Year>1999</Year><RecNum>175</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>175</REFNUM><YEAR>1999</YEAR><TITLE>Inhibitors of 5-lipoxygenase: a therapeutic potential yet to be fully realized?</TITLE><SECONDARY_TITLE>European Journal of Medicinal Chemistry</SECONDARY_TITLE><VOLUME>34</VOLUME><NUMBER>9</NUMBER><PAGES>671-685</PAGES><DATE>1999/9</DATE><AUTHORS><AUTHOR>Young, Robert N.</AUTHOR></AUTHORS><URL> activating protein</KEYWORD><KEYWORD>asthma</KEYWORD><KEYWORD>inflammation Inhibition of leukotriene biosynthesis has been extensively studied as a potential for the development of novel therapies for inflammation and respiratory diseases and, in particular, for asthma. Many compounds have been identified which inhibit the key enzyme, 5-lipoxygenase. Four distinct classes of compounds have been identified, namely, (1) redox inhibitors (alternative substrates), (2) iron chelating inhibitors, (3) competitive reversible inhibitors, and (4) inhibitors of the 5-lipoxygenase activating protein. Experience over the past two decades with redox inhibitors has been disappointing and although a number of potent compounds have been identified, they have often been associated with ancillary toxicity and non-specificity due to their redox activity. Iron chelating inhibitors have been more successful and one compound, Zileuton&#xAE;, has reached the market. However, more potent analogues have often encountered toxicity problems. Competitive inhibitors have been identified by a number of research groups but, as yet, none has been successful. Inhibitors of the 5-lipoxygenase activating protein (FLAP) have been identified and compounds such as MK-0591 and BaY-X-1005 have shown efficacy in asthma trials. To date, however, no clear advantage for inhibitors of lipoxygenase has been demonstrated relative to the leukotriene D4 receptor antagonists such as Singulair&#xAE; and Accolate&#xAE;.</KEYWORD></KEYWORDS><LABEL><styles></styles>Anti-inflammatory models</LABEL></MDL></Cite></EndNote>(Young 1999). Cytoxic assayP388D1 mouse lymphoblast cells and A375 human skin malignant melanoma cells were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA). Cell culture media was obtained from Invitrogen (Invitrogen Corporation, Carlsbad, CA, USA) and horse and foetal bovine sera from JRH Biosciences, Lenexa, KA, USA. Cell culture 96-well plates were obtained from Nunc (Thermo Fisher Scientific, Roskilde, Denmark). ATPLiteTM assay kits were obtained from PerkinElmer Bioscience (PerkinElmer Life and Analytical Sciences Pty. Ltd., Rowville Victoria).The P388D1 cells were routinely grown at 37 C, 5% CO2, in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% horse sera with 2mM L-glutamine, 100U/mL penicillin, and 100g/mL streptomycin. The A375 cells were routinely grown at 37 C, 5% CO2/95% air in DMEM containing 10% foetal bovine sera with 2mM L-glutamine, 100U/mL penicillin, and 100g/mL streptomycin. Proliferation assayThe purpose of this procedure was to investigate the potential of leaf extracts of D. viscosa to stimulate the growth of fibroblasts, using a colorimetric assay. Alamar Blue is a redox indicator that exhibits colorimetric and fluorescence changes upon exposure to cellular metabolic reduction. Cell proliferation induces chemical reduction of the media, resulting in a change of the colour of the indicator from blue to red, and the change was determined with the use of a spectrophotometer. Changes in cell numbers were assessed by measuring absorbance at 570nm and 600nm (Ansar Ahmed et. al. 1994; Biosource International 2005). PromoCell fibroblast growth medium kit (C-23120) and supplement pack were purchased from Banksia Scientific Pty. Ltd. (Bulimba, Qld.). After combining one 10mL supplement pack in 500mL of the basal medium the concentration of growth factors were: fetal calf serum (2%w/v) insulin (5μg/mL) and basic fibroblast factor (1ng/mL). Phosphate buffered saline (PBS), penicillin/ streptomycin solution, trypan blue solution was purchased from Sigma Aldrich Pty. Ltd., Castle Hill NSW. Trypsin/ EDTA and Trypsin/ neutraliser solution were purchased from Cascade Biologics (Oregon, USA). AlamarBlue was purchased from Biosource International (California, USA). PromoCell Normal Human Dermal Fibroblasts (NDHF) were purchased from Banksia Scientific Pty. Ltd. The number of viable cells was determined with a haemacytometer and a Leica DMIL inverse microscope. The culture was incubated in a Sanyo MCO-20AIC 37°C, 5% CO2/95% air humidified cell culture incubator. The supplemented growth media was replaced every second day until the fibroblasts approached confluence. When the culture neared confluence, standard subculturing procedures were followed. In the explant procedure, trypsinised fibroblasts were suspended in supplemented growth media dispensed from centrifugal tubes to seed each well of a 96-well flat-bottomed microplate in preparation for the proliferation assay. Cells were mixed thoroughly with a pasteur pipette for even distribution during the seeding procedure, at an approximate concentration of 4 x 103 cells/well. When seeding was complete, the microplate was incubated at 30°C, 5% CO2/95% air humidified cell culture incubator for 24 hours. An Alamar Blue solution was prepared according to the manufacturer’s instructions, to achieve a final concentration in the well of 2% v/v. Serial dilutions of sample extracts (10μL and 50μL) 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 less than 50% confluence were exposed to different concentrations of D. viscosa extracts. The control blanks consisted of fibroblast culture media with extraction solvents but without the addition of D. viscosa extracts. Since cell proliferation is indicated by Alamar Blue reduction, actual proliferation rates may be determined by comparing the reduction of the indicator by treated fibroblasts with that of untreated control wells (ie solvent blanks). The percentage difference in reduction is determined by an equation incorporating the molar extinction value for oxidation of Alamar Blue following the manufacturer’s protocol (Biosource International 2005). StatisticsWherever possible, analyses were carried out with five or more samples and their means reported. Data collected was subjected to the Student’s t-test (two data sets) or the one-way ANOVA for three or more data sets (p <0.05). When the ANOVA yielded significance, the Tukey-Kramer HSD test for multiple comparisons at the 5% significance level (95% confidence interval) was used to test the differences between all pairs of means.For fibroblast proliferation measurements, data was subjected to multivariate ANOVA analysis, treating the different time intervals as repeated measures due to the fact the same specimens were monitored on each occasion over time. All ANOVA and post-hoc tests were performed using JMP version 5.1 software.ResultsAntioxidant assayD. viscosa aqueous and ethanolic extracts exerted potent inhibition of DHR oxidation by AAPH induced peroxyl radicals, whether measured as percentage of oxidation inhibition compared to control or as Trolox equivalents (TE). However, aqueous extractions had notably higher levels of oxidation inhibition compared to samples prepared from ethanol or aqueous/ethanol solvent mixtures. Moreover, when extracts prepared using a spectrum of aqueous:ethanol ratios were compared, it was found that the 100% and 25% ethanol extracts were significantly more potent than the 45% and 60% ethanol extracts, but still significantly less potent than the aqueous extracts (Table 1). Oxidation inhibition levels by ascending concentrations of aqueous extracts are displayed in Figure 2. Table 1. Comparison of antioxidant activity of D. viscosa 10mg/mL ethanol extracts according to extraction solvent. % EthanolnOxidation inhibition (% of controls)* TE (μM)* 04**77.9 ± 1.136.8 ±0.625456.7 ± 8.7 a9.3 ± 5.5 45433.7 ± 9.8 b 3.1± 1.9 60427.3 ± 8.8 b 2.0 ± 0.9100455.7 ± 8.4 a 9.3 ± 3.8*values are mean ±SD. No SD is shown for 0% ethanol sample. **This value was arrived at by combining two experimentsValues not sharing a superscript indicate significant differences of mean oxidation inhibition as determined by Tukey-Kramer HSD comparisons (p<0.05)Figure 2. Dose response for D. viscosa aqueous samples (log10 concentration) against inhibition of AAPH oxidation by DHR, following induction by peroxyl radicals.. n = five separate extractions of D. viscosa leaves. r2=0.911, y=12.112x+71.187CytotoxicityD. viscosa aqueous extracts demonstrated no cytotoxicity, while methanol extracts were cytotoxic at high concentrations only (100μg/mL). Acetone extracts however were cytotoxic at 33μg/mL and 100μg/mL, while at low concentrations (11μg/mL and below) it appeared that methanol extracts demonstrated a proliferative effect on A375 cells. It was not possible to establish IC50 values due to the lack of toxicity of some of the samples. Lipoxygenase inhibition assayThe ethanol extract was the only preparation to show any LOX inhibition (Figure 3); when 45% ethanol and aqueous D. viscosa extracts were tested in this assay they failed to show any inhibition (data not shown).Figure 3. Linearity of response between different concentrations of D. viscosa ethanol extract and LOX inhibition. r2 = 0.89, p<0.01, y = 0.0568x + 6.4701Fibroblast proliferationThe fibroblast assay demonstrated proliferative effects on the growth of human fibroblasts for aqueous, 45% ethanol and 100% ethanol D. viscosa extracts. The fibroblasts incubated with the 45% ethanol extract consistently achieved proliferation rates which were higher than those grown with the 100% ethanol extract over the 48 hours (Figure 4). With Alamar Blue relative reduction rates (relative to their respective solvents only) at 24 hours of 204% and 364% and at 48 hours of 277 % and 532% for the 100% and 45% ethanol extracts respectively.Figure 4. The effects of D. viscosa 100% and 45% ethanol extract on fibroblast proliferation. The relative reduction of Alamar Blue by fibroblasts grown in the presence of D. viscosa extract with 100% ethanol and 45% ethanol at concentrations of 50μg/mL was expressed as a percentage relative to cells incubated with solvent only and followed over 48 hours. Overall these studies indicate D. viscosa has substantial antioxidant, LOX inhibition and fibroblast proliferation activities in vitro with minimal cytotoxic effects, however the degree of activity is dependent upon the extraction solvent used. Discussion D. viscosa preparations have been used in Australia and elsewhere for wound healing, but clinical and experimental evidence is lacking. It is possible the wound healing activity derives from a primary action associated with proliferation or migration of fibroblasts to the wound, and/or is secondary to other actions such as anti-inflammatory, antioxidant or antimicrobial processes. In the antioxidant studies a distinct pattern of activity relative to the extraction solvent used was demonstrated, with 100% aqueous extracts in the highest range, 100% and 25% ethanol extracts in the mid-potency range, while those prepared with 45% and 60% ethanol provided the weakest activity. The reason for this phenomenon may be related to phytochemical differences in the extracts, since flavonoid content as assessed by HPLC with diode-array detection have been shown to vary significantly in accordance with the ethanol content of the extraction solvent (unpublished data, Pengelly 2009).All D. viscosa extracts tested were effective in enhancing fibroblast proliferation, however the aqueous and 45% ethanol extracts provided stronger inhibition when compared to 100% ethanol extracts. The findings from the cytotoxicity assays provide some support for these results, despite the differences in extraction solvents used. Aqueous extracts were not cytotoxic at 100μg/mL, however the acetone and methanol extracts exhibited considerable cytotoxic activity at 33μg/mL and 100μg/mL respectively. Therefore, acetone and methanol may be best avoided for preparation of D. viscosa phytomedicines. This finding is in line with the general philosophy for herbal medicine manufacturing practice which promotes the use of water and ethanol as the safest solvents (Etkin 1988; Bone 2003). The results support other studies that demonstrate low toxicity and skin irritancy for D. viscosa (Teshome et al 2010).The antioxidant activity demonstrated for D. viscosa may contribute to its’ potential as an anti-inflammatory or wound healing agent ADDIN EN.CITE <EndNote><Cite><Author>van Hien</Author><Year>1997</Year><RecNum>308</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>308</REFNUM><AUTHORS><AUTHOR>van Hien, T.</AUTHOR><AUTHOR>Margaret, A </AUTHOR><AUTHOR>Hughes, G. W. </AUTHOR><AUTHOR>Cherry, C.</AUTHOR></AUTHORS><YEAR>1997</YEAR><TITLE>In vitro studies on the antioxidant and growth stimulatory activities of a polyphenolic extract from Cudrania cochinchinensis used in the treatment of wounds in Vietnam</TITLE><SECONDARY_TITLE>Wound Repair and Regeneration</SECONDARY_TITLE><VOLUME>5</VOLUME><NUMBER>2</NUMBER><PAGES>159-167</PAGES><LABEL>Wound &amp; injury healing</LABEL></MDL></Cite><Cite><Author>Thang</Author><Year>2001</Year><RecNum>306</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>306</REFNUM><YEAR>2001</YEAR><TITLE>Anti-oxidant effects of the extracts from the leaves of Chromolaena odorata on human dermal fibroblasts and epidermal keratinocytes against hydrogen peroxide and hypoxanthine-xanthine oxidase induced damage</TITLE><SECONDARY_TITLE>Burns</SECONDARY_TITLE><VOLUME>27</VOLUME><NUMBER>4</NUMBER><PAGES>319-327</PAGES><DATE>2001/6/1</DATE><AUTHORS><AUTHOR>Thang, Phan Toan</AUTHOR><AUTHOR>Patrick, See</AUTHOR><AUTHOR>Teik, Lee Seng</AUTHOR><AUTHOR>Yung, Chan Sui</AUTHOR></AUTHORS><URL> &amp; injury healing</LABEL></MDL></Cite></EndNote>(van Hien et al. 1997; Thang et al. 2001). 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). Peroxyl radicals cause direct damage to DNA and deplete endogenous antioxidants ADDIN EN.CITE <EndNote><Cite><Author>Halliwell</Author><Year>1995</Year><RecNum>35</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>35</REFNUM><YEAR>1995</YEAR><TITLE>The characterization of antioxidants</TITLE><SECONDARY_TITLE>Food and Chemical Toxicology</SECONDARY_TITLE><VOLUME>33</VOLUME><NUMBER>7</NUMBER><PAGES>601-617</PAGES><DATE>1995/7</DATE><AUTHORS><AUTHOR>Halliwell, B.</AUTHOR><AUTHOR>Aeschbach, R.</AUTHOR><AUTHOR>Loliger, J.</AUTHOR><AUTHOR>Aruoma, O. I.</AUTHOR></AUTHORS><URL> models</LABEL></MDL></Cite></EndNote>(Halliwell et al. 1995). Therefore the present demonstration of free radical oxidation inhibition in D. viscosa extracts could offer a mechanism for addressing inflammatory disorders in humans. Although not tested during this project, documented analgesic (Amabeoku et al. 2001), antipruritic (Pearman 2000; van Wyk 2008) and antimicrobial (Teffo, Aderogba and Eloff 2010) properties of D. viscosa could enhance the antioxidant and fibroblast proliferation activities reported here, providing a sound rationale for further promoting the use of this traditional plant medicine as a healer of wounds and treatment for inflammatory skin disorders. In conclusion, traditional use of Dodonaea viscosa subsp. angustifolia leaf extracts for localised inflammation and wound-healing was further validated by a combination of in vitro studies. Further investigations are needed to establish likely mechanisms of action.as well as the most effective solvents and extraction methods. The combination of findings from recent in vivo studies and the current investigations provide further impetus to have the species considered for listing on the Australian Register of Therapeutic Goods, potentially paving the way for studies on humans.AcknowledgementThe paper derives from my PhD project at the University of Newcastle, Australia. It received financial support from Southern Cross Herbal Extracts and the Rural Industry Research and Development Corporation. I am grateful to my supervisors Dr. Paul Roach and Dr. Mark Lucock for reviewing this paper and providing constructive comments and suggestions. ReferencesAboriginal Community of the Northern Territory of Australia 1993. Traditional Aboriginal Medicines, Conservation Commission of the Northern territory, Darwin.Amabeoku, G J, Eagles P, Scott G, Mayeng I, Springfield E. 2001. Analgesic and antipyretic effects of Dodonaea angustifolia and Salvia africana-lutea. J. Ethnopharmacology, 75: 117-124.Arun M, Asha VV. 2008. 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Inhibitors of 5-lipoxygenase: a therapeutic potential yet to be fully realized? European J. Medicinal Chemistry 34: 671-685. ................
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