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Antioxidant capacity and phenolic compounds in commercially grown native
Australian herbs and spices
Running title: Ethnic Australian herbs and spices
Izabela Konczak*, Dimitrios Zabaras, Matthew Dunstan, Patricia Aguas
CSIRO Food and Nutritional Sciences, PO Box 52, North Ryde, NSW 1670, AUSTRALIA
*Corresponding author. Tel.: +61 2 94908563. Fax: +61 2 9490 8499, e-mail:
[email protected]
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Abstract.
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The antioxidant capacities and phenolic composition in six native, commercially
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grown, Australian herbs and spices were investigated. Tasmannia pepper leaf,
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followed by anise myrtle and lemon myrtle contained the highest levels of total
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phenolics (TP; 102.1; 55.9 and 31.4 mg gallic acid equivalents (GAE)/g dry weight
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(DW), respectively). Tasmannia pepper leaf exhibited the highest oxygen radical
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absorbance capacity (ORAC assay) followed by lemon myrtle and anise myrtle. Anise
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myrtle exhibited the highest total reducing capacity [TRC; Ferric Reducing
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Antioxidant Power (FRAP) assay], followed by Tasmannia pepper leaf and lemon
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myrtle. Australian bush tomato, with TP content of 12.4±0.9 mg GAE/gDW and TRC
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of 206.2µMol Fe+2/gDW, resembled the Chinese Barbary Wolfberry fruit. The TP
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content of Tasmannia pepper berry (16.86 mg GAE/gDW) was similar to that of black
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pepper, but it’s TRC was 25% lower. Cinnamic acids and flavonoids, tentatively
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identified by mass spectrometry, were identified as the main sources of antioxidant
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activities.
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1. Introduction
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Antioxidant properties of spices have been recognized since 1952, when Chipault and
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co-workers demonstrated that leaves of rosemary and sage effectively increased the
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antioxidant capacity of foods and the effect depended on food matrices (Chipault,
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Mizuno, Hawkins & Lundberg, 1952). Research over the last decade has delivered a
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vast amount of data indicating possible prevention of chronic diseases by antioxidant
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phytochemicals in food (Liu, 2004). Studies on culinary and medicinal herbs
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identified their superior antioxidant activity to berries, other fruits, vegetables, and
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nuts (Zheng & Wang, 2001; Wojdylo, Oszmianski & Czemerys, 2007). The use of
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herbs and spices in food is steadily increasing (Sloan, 2005), especially since
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consumers have questioned the use of the synthetic antioxidants butylated
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hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) in food products
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(Madsen & Bertelsen, 1995).
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The rich Australian flora, comprising over 25,000 native plants which have developed
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in geographical isolation from the northern hemisphere, offer a number of attractive
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edible species that have been used as a food and medicine by the native population for
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thousands of years (Cooper, 2004). Early colonial Australians have incorporated a
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number of these edible species into their daily diet. Descriptions of native edible
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plants by ethnobotanists existed at the end of the 19th century (Maiden, 1889). To
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date, a number of native herbs and spices has entered commercial production in
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Australia and established themselves as valuable components of the ‘Australian
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cuisine’, bringing original flavors and desirable sensory properties (Hodgson &
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Wahlqvist, 1992).
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Among the most popular herbs and spices is Tasmannia pepper (Tasmannia
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lanceolata, Winteraceae). The plant grows into an attractive shrub up to 5 metres high
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with dark green leaves and distinctive crimson stems. The fruit is a black, aromatic
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berry about 6-7 mm in diameter (Drager, Garland & Menary, 1998). The leaves, used
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as a herb, and berries, used as a spice, are now used to give a ‘wild, natural and spicy’
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taste to foods of the Australian native cuisine.
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Lemon myrtle (Backhousia citriodora) of the Myrtaceae family, a native to
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subtropical rainforests of Queensland, is probably the most commercialized native
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spice with thousands of trees now under cultivation. The evergreen tree bears glossy
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green, lanceolate leaves that are 5-12 cm long and 1.5-2.5 cm broad and creamy-white
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flowers, 5-7 mm diameter, produced from summer through to autumn. Highly valued
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for their strong lemon flavor, the leaf and flowers are used in tea blends and
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beverages, dairy, biscuits, breads, confectionery, pasta, syrups, liqueurs, flavoured
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oils, packaged fish (salmon), dipping and simmer sauces. It can also be used as a
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lemon-flavor replacement in milk-based foods, such as cheesecake, lemon flavored
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ice-cream, and sorbet without the curdling problem associated with lemon fruit
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acidity. The source of intensive lemon flavour is citral that makes typically 95% of the
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steam distilled lemon myrtle oil (Southwell, 1996).
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Anise myrtle (Syzygium anisatum, Myrtaceae) is a rare Australian rainforest tree from
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the north-east NSW and Queensland region. The leaf, used as a herb, contains 79.4 to
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90% of (E)-anethole and 4.4 to 10.1% methyl chavicol (Southwell, Russel,
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Birmingham, & Brophy, 1996). The leaves can be used fresh or dry ground. They
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provide an aniseed flavor to sweet and savory dishes as well as to cosmetics.
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Wattle trees (Acacia sp.) are the dominant trees in central Australia and their seeds are
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consumed by the Aboriginal population as a staple food. Wattle seeds are among the
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commercially available native spices and among them the seeds of ‘Elegant Wattle’
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(Acacia victoriae) are regarded by many as the food industry standard. Other
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commonly traded species include: Acacia colei, A. coriacea, Golden Wattle (A.
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pycnantha), Sandplain Wattle (A. murrayana), Silver Wattle (A. retinodes) and
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Coastal Wattle (A. sophorae) (Cribb, Latham & Ryder, 2005). The roasted and ground
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seeds have a ‘nutty’ flavour and are included into baked goods, flour mixes, mustards,
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dressings, sweet sauces and beverages. They are high in protein and have a low
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glycemic index and were proposed for inclusion in diabetic and other specialty diets
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(Cribb, Latham & Ryder, 2005). Triterpenoid saponins isolated from Acacia victoriae
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seeds were identified to inhibit activation of nuclear factor – kappa B (NFKB), and
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possible suppression of the development of malignant cells (Haridas, Arntzen &
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Gutterman, 2001). Laboratory testing and human trials on the edible Acacia species
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have shown that the seed is highly nutritious and safe to eat (CSIRO Media release,
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98/213, Sept. 9, 1998).
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Bush tomatoes (Solanum centrale, Solanaceae), a native spice also known as the
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desert raisin, grow in the Australian Central Desert. The plant is small (30 cm tall) and
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produces yellow berry fruit. The fruit resembles the Chinese wolfberry fruit (Lycium
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barbarum L., Solanaceae), also known as ‘goji berry’. The fruits, when left to dry on
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the plant, develop an intense, earthy-tomato and caramel flavour of great piquancy
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and pungency. Two other species, S. chippendalei and S. ellipticum also produce
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edible fruit and are of interest to the native food industry (Cribb, Latham & Ryder,
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2005).
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All of the above mentioned herbs and spices are currently in commercial production
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in Australia. However, to date the knowledge about their phytochemical composition
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and antioxidant capacity is limited. Miller and co-workers (Miller, James &
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Maggiore, 1993) have published general compositional data of about 500 different
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indigenous Australian foods. This includes information on the content of water,
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protein, fat, carbohydrates, selected vitamins and minerals. The study revealed that
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most of the ethnic Australian foods have similar composition to common foods in the
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same category. The objective of our study was to evaluate selected species that are of
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importance to the Australian Native Food Industry, for the presence of
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phytochemicals important to human health, and to compare them to the levels of these
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phytochemicals in traditionally consumed foods.
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It needs to be mentioned that the results obtained in this study originate from a single
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lot of samples produced during one vegetative season using plant sources selected by
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the industry. Variations in the levels of phenolic compounds and antioxidant
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capacities arising from the genetic diversity and the environmental factors were not
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evaluated in this study.
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2. Materials and Methods
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2.1. Plant Material
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Six samples, selected by the Australian Native Food Industry Ltd. (ANFIL), were
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used. Samples of Tasmannia pepper (Tasmannia lanceolata R. Br.,) leaves and
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berries, were supplied by the company Diemen Pepper (Tasmania, Australia). Anise
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myrtle (Syzygium anisatum (Vickery, Craven & Biffen) and lemon myrtle
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(Backhousia citriodora F.Muell) were obtained from Australian Rainforest Products
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(NSW, Australia). Bush tomato (Solanum centrale J.M.Black) and wattle seeds
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(Acacia sp.) were supplied by the Outback Pride company (Reedy Creek, South
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Australia). The dry samples were finely ground upon arrival.
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2.2. Extraction of hydrophilic compounds
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An aliquot (250 mg) of the ground sample was extracted with 5 mL of 80% aqueous
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methanol/1.0% HCl (v/v) under a nitrogen atmosphere to prevent oxidation. The
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samples were sonicated for 10 minutes, centrifuged (10 min, 5000 rpm), and the
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supernatant collected. The pellet was re-extracted two more times. Aliquots of the
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combined supernatants (15 mL) were filtered with a 13 mm x 0.45 µm
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polytetrafluoroethylene (PTFE) membrane, and stored at 0.5°C under nitrogen until
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analyzed. The extraction was carried out in triplicate for each sample. The analysis
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was conducted within 3 days.
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2.3. Extraction of lipophilic compounds
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An aliquot (250 mg) of the ground sample was extracted with 10 mL of cold acetone
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(4°C). The samples were shaken for 20 minutes, centrifuged (10 min, 2500 rpm), and
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the supernatants were collected. The pellet was re-extracted two more times. Freshly
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prepared aliquots of the combined supernatants (30 mL) were filtered with a 13 mm x
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0.45 µm PTFE filter membrane and immediately analyzed. The extraction was carried
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out in triplicate for each sample.
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2.4. The total phenolic content (TP)
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The total phenolic content was determined using the Folin-Ciocalteu assay (Singleton
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& Rossi, 1965). Diluted extracts were directly assayed at 600 nm with gallic acid as a
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standard. The analysis was conducted in triplicate and the results were corrected for
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vitamin C. Results were expressed as milligrams of total phenolics (gallic acid
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equivalents) per gram dry weight (mg GAE/g DW). Measurements were done in
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microplates using a microplate reader model Multiscan RC, version 4 (Labsystems,
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Finland) operated by the DeltaSoft3 program (Elisa Analysis for the Macintosh with
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interference for the Multiscan Microplate Readers, BioMetallics, Inc., 1995).
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2.5. Oxygen Radical Absorbance Capacity (ORAC) assay
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ORAC-H (hydrophilic compounds). The assay for oxygen radical scavenging capacity
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was conducted according to Prior, Wu & Schaich (2005) and Ou, Hampsch-Woodill
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& Prior (2001). The samples (in triplicate) were mixed with a fluorescein (15 nM)
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solution and a solution of 2,2’-azobis-(2-methylpropionamidine) dihydrochloride
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(AAPH, 360 mM) both in phosphate buffered saline (PBS, 75 mM, pH 7.0). Both
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AAPH and PBS buffer were warmed to 37°C prior to use. The fluorescence was
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recorded until it reached zero (excitation wavelength 495 nm, emission wavelength
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515 nm) in a Varian Cary Eclipse Fluorescence Spectrophotometer equipped with an
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automatic thermostatic autocell holder at 37 °C. A calibration curve was constructed
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daily by plotting the calculated differences of area under the fluorescein decay curve
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between the blank and the sample for a series of standards of Trolox solutions in the
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range of 6.25 - 75 µg/L. The results were expressed as μmol Trolox equivalents per
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100 gram dry weight (µmol Trolox Eq./100g DW).
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ORAC-L (lipophilic compounds). All the reagents were prepared using 75 mMol
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phosphate buffer (pH 7.4) as described for the ORAC-H assay. Samples and Trolox
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standards were made in 7% (w/v) randomly methylated β-cyclodextrin (RMCD)
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solvent to ensure solubility of the lipophilic antioxidant in the reaction mixture
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(Huang, Hampsch-Woodill, Flanagan & Deemer, 2002). The 7% RMCD solvent was
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made in a 50% acetone-water mixture (v/v) and was shaken for 1 hour at room
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temperature on an orbital shaker at 200 rpm prior to use. The sample solution was
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ready for analysis after further dilution with 7% RMCD. The measurements were
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conducted as described for the ORAC-H method. ORAC-T represents the sum of
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ORAC-H and ORAC-L.
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2.6. FRAP (Ferric Reducing Antioxidant Power) assay
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The assay was conducted according to Benzie & Strain (1996) with minor
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modifications. Thirty µL of water and 10 µL extracts were mixed with 200 µL FRAP
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reagent consisting of ferric chloride and 2,4,6-tripyridyl-s-triazine (TPTZ). The
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absorbance was measured after 4 min at 600 nm. The reducing capacity was
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calculated using the absorbance difference between sample and blank and a further
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parallel Fe(II) standard solution. Results were expressed as micromoles of Fe2+ per
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gram dry weight (µmol Fe2+/g DW). Measurements (in triplicate) were done in
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microplates as described for total phenolics.
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2.7. Analysis of phenolic compounds by high performance liquid
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chromatography-diode array detector (HPLC-DAD) and liquid
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chromatography-photodiode array-mass spectrometry (LC-PDA-MS/MS)
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HPLC-DAD analysis. Quantification of phenolic compounds in extracts was carried
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out using a High Performance Liquid Chromatography system that consisted of two
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LC-10AD pumps, SPD-M10A diode array detector (DAD), CTO-10AS column oven,
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DGU-12A degasser, SIL-10AD auto-injector and SCL-10A system controller
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(Shimadzu Co., Kyoto, Japan) equipped with a 250 x 4.6 mm i.d., 5 µ Luna C18(2)
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column (Phenomenex, NSW, Australia). The following solvents in water with a flow
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rate of 1.0 mL/min were used: A, 0.5% triflouroacetic acid (TFA) in water and B,
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95% acetonitrile and 0.5% TFA in water. The elution profile was a linear gradient
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elution for B of 10% over 10 minutes followed by an increase to 50% over 45 min,
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and than to 80% over 15 minutes. The column was washed with 100% solvent B for
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10 minutes. Analytical HPLC was run at 25°C and monitored at 280 nm
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(hydroxybenzoic acids and flavanols), 326 nm (hydroxycinnamic acids, stilbenes),
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370 nm (flavonols) and 520 nm (anthocyanins). Hydroxybenzoic acids and flavanols
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were quantified as gallic acid equivalents (GA Eq.), cinnamic acids were quantified as
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chlorogenic acid equivalents (CHA Eq.), flavonols and stilbenes were quantified as
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rutin equivalents (R Eq.) and anthocyanin compounds were quantified as cyanidin 3-
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glucoside equivalents (C3G Eq.). The results are presented per gram of dry weight
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(e.g. mg C3G Eq/g DW).
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LC-PDA-MS/MS analysis. LC-PDA-MS/MS analysis was carried out on a Quantum
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triple stage quadrupole (TSQ) mass spectrometer (ThermoFinnigan, NSW, Australia)
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equipped with a quaternary solvent delivery system, a column oven, a photo-diode
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array (PDA) detector and an autosampler. An aliquot (20 μl) from each extract was
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chromatographed on a Luna C18(2) analytical column (150 mm x 2.1 mm, 5 μm
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particle size), (Phenomenex), which was heated to 30◦C in an oven. The mobile phase
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consisted of 0.5% formic acid in water (A) and 0.5% formic acid in acetonitrile (B) at
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the rate of 220 µL/min. A linear gradient was used (0% B to 100% B over 40 min).
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Ions were generated using an electrospray source in the positive or negative mode
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under conditions set following optimisation using solutions of cyanidin-3-glucoside,
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chlorogenic acid and rutin. The PDA was monitoring signals at 520, 370, 320 and 280
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nm. MS experiments in the full scan (precursor and product-specific) and the selected
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reaction monitoring (SRM) mode were carried out.
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2.8. Extraction and analysis of vitamin C
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Vitamin C was extracted from powdered samples and stabilised using 4.5% meta-
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phosphoric acid according to Vazquez-Oderiz, Vazques_Blanco, Lopez-Hernandez,
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Simal-Lozano & Romero-Rodriguez (1994). An aliquot (50 mg) of each sample was
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mixed with 1500 μL of 4.5% m-H3PO4, vortexed and sonicated for 5 minutes to
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enhance the extraction process. Subsequently, the samples were centrifuged (5 min,
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12000 rpm) to remove solid particles. The supernatants were collected and the
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extraction was repeated two more times. The supernatants were pooled together (4.5
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mL). The extracts were prepared and analysed in triplicate. Representative samples
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(10 μL, three replicates) were injected into HPLC (equipment details as above).
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Vitamin C was separated under isocratic conditions using water acidified with
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sulphuric acid to pH 2.2 following the method of Vazquez-Oderiz, Vazquez-Blanco,
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Lopez-Hernandez, Simal-Lozano & Romero-Rodriguez (1994). Detection was carried
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out at 245 nm at a flow rate of 1.0 mL/min. Vitamin C was identified by comparing
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the retention time and characteristic UV-VIS spectra with those of synthetic L-
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ascorbic acid (Sigma, Sydney, Australia). The results were quantified using an L-
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ascorbic acid calibration curve and calculated as micromoles per mL (µmol/mL). The
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limit of detection was 1 µg/mL.
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3.0. Results and Discussion
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3.1. Total phenolic content
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The Folin-Ciocalteu procedure has been proposed to rapidly estimate the level of total
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phenolics in foods and supplements (Prior, Wu & Schaich, 2005). The levels of
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phenolic compounds in the evaluated species varied significantly from 0.76 to 102 mg
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GAE/gDW (Table 1). The richest source of phenolic compounds was Tasmannia
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pepper leaf, followed by anise myrtle and lemon myrtle. Total phenolic content of
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these Australian herbs was compared with that of basil leaf (Ocimum basilicum L.), a
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Mediterranean herb widely consumed around the world. The level of phenolic
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compounds in 23 accessions of basil grown in Iran varied from 23.0 to 65.5 mg GA
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E/gDW (Javanmardi, Stushnoff, Locke & Vivanco, 2003). Subsequently, the content
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of phenolic compounds in Tasmannia pepper leaf is 2- to 4-fold that of basil leaf.
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Total phenolic content of two other Australian herbs, anise myrtle and lemon myrtle,
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are comparable to that of basil. Anise myrtle and lemon myrtle are also comparable to
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Chinese star anise and nutmeg (53.89 ± 0.82 and 37.26 ± 0.66 mg GAE/gDW,
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respectively) (Liu, Qiu, Ding & Yao, 2008). These three Australian herbs also contain
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more phenolic compounds than peppermint leaf (13.17±0.04 mg GAE/gDW), the leaf
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of perilla widely used in Japan (11.3±0.16 mg GAE/gDW) and mulberry leaf used in
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China (25.22 ± 0.36) (Liu, Qiu, Ding & Yao, 2008). They also contain more phenolic
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compounds than European camomile and thyme (12.7±0.7and 17.1±0.2 mg
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GAE/gDW, respectively) (Kahkonen et al., 1999). The levels of total phenolics in
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Tasmannia pepper leaf, anise myrtle and lemon myrtle are similar to the TP levels in
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maple leaf, silver birch leaf and needle of spruce (31.7±0.2; 38.4±1.0; 155.5±6.1 mg
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GAE/gDW, respectively) (Kahkonen et al., 1999).
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The phenolic content of Tasmannia pepper berry (Table 1) was comparable to that of
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black pepper (Piper nigrum) (17.16±0.11 mg GAE/gDW) and total phenolic content
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of Bush Tomato was identical with that of Chinese Barbary Wolfberry fruit (Lycium
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barbarum L.) (12.53±0.62 mg GAE/gDW) (Liu, Qiu, Ding & Yao, 2008). These
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levels of TP were within the same range as TP in selected Algerian medicinal plants
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(Djeridane, Yousfi, Nadjemi, Boutassouna, Stocker & Vidal, 2006). The seed of the
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wattle tree had the lowest content of phenolic compounds among the evaluated
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samples. It was similar to the TP of flax seed (0.8 ± 0.1 mg GAE/gDW) and
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approximately half of that of black sesame and peach kernel (1.34±0.01 and
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1.32±0.02mg GAE/gDW) (Kahkonen et al., 1999).
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3.2. Antioxidant capacity of fruits
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Total reducing capacity. Anise myrtle displayed the strongest total reducing capacity
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(TRC) as evaluated in the FRAP assay (Table 1), and was followed by the Tasmannia
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pepper leaf and lemon myrtle. The TRC of anise myrtle was similar to that of Chinese
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star anise (2.685±0.04 mMol Fe+2/gDW) and the TRC’s of Tasmannia pepper and
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lemon myrtle leaves were similar to that of perilla leaf and nutmeg (1.113±0.01 and
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1.255±0.04 mMol Fe+2/gDW, respectively) (Liu, Qiu, Ding & Yao, 2008). The TRC
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of Tasmannia pepper berry was 72.6% of that of black pepper (Liu, Qiu, Ding & Yao,
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2008). The TRC of the Australian bush tomato was identical to that of Chinese
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Barbary Wolfberry fruit (Lycium barbarum L.) (0.207±0.002 mMol Fe+2/gDW) (Liu,
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Qiu, Ding & Yao, 2008). In respect to the total phenolic content and the TRC the
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Australian bush tomato and the related Chinese wolfberry (or goji berry) displayed
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identical characteristics. Wattle seeds displayed very low TRC and were comparable
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to lotus seeds (0.010±0.00 mMol Fe+2/gDW) (Liu, Qiu, Ding & Yao, 2008).
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Oxygen radical scavenging capacity (ORAC). For the chemical estimation of
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antioxidants in foods the oxygen radical absorbance capacity assay has been proposed
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as the preferred assay with possible relevance to human physiology (Prior, Wu &
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Schaich, 2005). The highest ability to scavenge the oxygen free radicals was
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displayed by the leaf of Tasmannia pepper and was followed by other samples in the
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following order: lemon myrtle > anise myrtle > Tasmannia pepper berry > bush
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tomato > wattle seed. Most of the published research data on the antioxidant activity
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of edible plant sources as measured in the ORAC assay is presented as micromoles of
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Trolox equivalents per gram of fresh weight (µMol TE/gFW) (Zheng & Wang, 2001).
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Based on the literature we have recalculated the results for sweet basil presented by
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Zheng & Wang (2001) into µMol TE/gDW accepting that the dry weight content of
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sweet basil is 6.6% according to Di Cesare, Forni, Viscardi & Nani (2003). The result
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indicates that the antioxidant activity of sweet basil expressed as micromoles of
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Trolox equivalents per gram of dry weight would be approximately 216.2 µMol
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TE/gDW. Among the spices evaluated by Zheng and Wang the ‘sweet bay leaf’
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resembles the Tasmannia pepper leaf, lemon myrtle and anise myrtle. The moisture
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content of bay leaf is estimated to be 90 to 95%. Taking these values into account, we
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have calculated that commercially available dry product produced from the bay leaf,
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as evaluated by Zheng & Wang (2001) would contain between 40.2 to 80.4 mg
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GAE/gDW of total phenolics and would exhibit antioxidant activity in H-ORAC of
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317 to 634 µMol TE/gDW. This indicates that the Australian herbs mentioned above
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contain similar (lemon myrtle and anise myrtle) or a higher levels (Tasmannia pepper
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leaf) of phenolic compounds and possess superior radical scavenging ability in
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comparison to sweet basil and bay leaf.
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With the exception of lemon myrtle, the main source of oxygen radical absorbance
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capacity in the evaluated sources was the hydrophilic fraction, e.g. 86% and 95% for
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Tasmannia pepper leaf and anise myrtle, respectively (Table 1). In the case of lemon
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myrtle, the hydrophilic fraction contributed 56.2% and the lipophilic fraction 45.8%
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to the total oxygen radical absorbance capacity. The high values of ORAC-L are
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possibly due to the presence in lemon myrtle of an essential oil that displays a high
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antioxidant capacity (Ruberto & Baratta, 2000). The contribution of the lipophilic
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fraction to the total oxygen radical absorbance capacity in Tasmannia pepper leaf was
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14.0%, in Tasmannia pepper berry - 18.5%, Bush Tomato – 2.0% and wattle seed –
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13.2%.
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With the exception of anise myrtle, all samples evaluated in this study do not
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contain vitamin C (Table 1). We have examined a correlation between the levels of
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phenolic compounds and antioxidant capacity as obtained in FRAP and ORAC
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assays. The high value of correlation coefficients (Table 2) indicates that phenolic
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compounds are the major contributor to the antioxidant capacity of the hydrophilic
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extracts. These results were expected and they confirm results published by others
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(Zheng & Wang, 2001).
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3.3. Identification of major phenolic compounds
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Selected phenolic compounds in the Australian native herbs were separated and
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tentatively identified by using a reversed-phase HPLC and LC/MS (Table 3 and
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Figure 1). The major groups of phenolic compounds detected were: phenolic acids
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(benzoic and cinnamic) and flavonoids (flavonols, flavanones and anthocyanins). The
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same types of phenolic compounds were described earlier as the main sources of
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antioxidant activities in edible plants (Kahkonen et al., 1999).
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The phenolic composition of Tasmannia pepper berry consisted predominantly of
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cyanidin 3-rutinoside and cyanidin 3-glucoside, chlorogenic acid, rutin and quercetin
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(Table 3, Figure 1). The characteristic feature of these molecules is the ‘catechol’
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structure or presence of at least 2 HO- groups on a benzene ring that is responsible for
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the enhanced antioxidant properties of phenolic compounds (Rice-Evans, Miller &
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Paganga, 1996). The phenolic mixture of the Tasmannia pepper leaf comprised of the
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same compounds; however anthocyanins were present at a very low level (1.25 mg
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C3G/g DW) and the mixture was dominated by cinnamic acids. Chlorogenic acid was
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the major compound, making up about 3% of the sample’s dry weight. A similarly
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high concentration of chlorogenic acid has been identified in the leaf of white birch
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(Ossipov, Nurmi, Loponen, Haukioja & Philaja, 1996).
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The anise myrtle extract contained several components with an m/z 303 under positive
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electrospray ionisation (ESI). This could possibly indicate the presence of quercetin or
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hesperitin aglycone(s) in the extract. These components were responsible for the
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major peaks at 370 nm. The group included a rhamnoside, a pentoside, a hexoside
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and a rutinoside. Other detected components in minor amounts included myricetin and
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chlorogenic acid. Similarly, the major components in the lemon myrtle extract were
350
also found to contain an m/z 303 algycone under positive ESI. This aglycone
351
represents both hesperitin and quercetin (both exhibit an m/z of 303 in positive ESI)
352
and further identification is in progress to confirm the identity of this compound.
353
Major glycosides found were hexosides, a ramnoside, and a pentoside. Traces of
354
rutin/hesperidin and naringenin rutinoside were also found. Based on mass
355
spectrometric evidence, Bush Tomato is likely to contain quercetin rutinosides, a
356
quercetin hexoside, and a kaempferol or luteolin hexoside. Minor amounts of
357
chlorogenic, caffeic, ferulic, coumaric and hydroxybenzoic acids were also detected in
358
the extract (Table 3). Similarly to Bush Tomato, phenolic compounds were present at
359
minute quantities in the extract of wattle seed. Compounds detected included rutin,
360
quercetin and hexosides containing an m/z 285 under negative (m/z 287 under
361
positive) ESI being indicative of kaempferol or luteolin aglycones. Trace levels of
362
chlorogenic acid were also found.
363
The present study showed high diversity in the levels and composition of phenolic
364
compounds in the evaluated Australian herbs. Among them Tasmannia pepper leaf,
365
anise myrtle and lemon myrtle were identified as superior sources of antioxidant
366
capacities. Phenolic compounds, especially cinnamic acids and flavonoids, were
367
identified as the major sources of antioxidant capacities. In the case of lemon myrtle,
368
lipophilic compounds also contributed significantly towards the antioxidant activity.
15
369
The phenolic composition of Tasmannia pepper leaf that displayed the highest
370
antioxidant capacity consisted predominantly of chlorogenic acid which equalled to
371
approximately 3% of the sample’s dry weight. The other major phenolic compounds
372
of both the leaf and berry of Tasmannia pepper were cyanidin 3-glucoside and
373
cyanidin 3-rutinoside, rutin and quercetin. The lowest antioxidant activity was
374
exhibited by the Bush Tomato and wattle seeds that contained cinnamic acids at very
375
low levels and only traces of flavonoid compounds.
376
377
4. Conclusions
378
The native Australian herbs and spices evaluated represent a new rich source of
379
antioxidant compounds of a phenolic nature: benzoic / cinnamic acids and flavonoids.
380
With respect to the composition of phenolic compounds Australian herbs resemble
381
other commonly used herbs. With the observed multiplicity of phytochemicals the
382
native herbs may contribute greatly to diversify and enhance the health-maintaining
383
properties of the Australian daily diet.
384
Acknowledgements
385
Financial support by the Rural Industries Research and Development Corporation
386
(RIRDC) and the Australian Native Food Industries Ltd. (ANFIL) towards this
387
research is gratefully acknowledged.
388
References
389
390
391
392
Benzie, I.F.F., Strain, J.J. (1996) The ferric reducing ability of plasma (FRAP) as a measurement
of “antioxidant power”: the FRAP assay. Analytical Biochemistry, 239: 70-76.
Chipault, J.R., Mizuno, G.R., Hawkins, J.M. Lundberg, W.O. (1952) The antioxidant properties of
natural spices. Food Research, 17: 46-55.
16
393
394
395
Cooper, W. (2004). Fruits of the Australian tropical forest. Nokomis Editions Pty Ltd., Melbourne,
Victoria, Australia, p. 616.
Cribb, J., Latham, Y., Ryder, M. (2005) Indigenous Australian foods for the global palate.
396
Terrain.org.
397
(http://www.terrain.org/articles/16/cribb_latham_ryder.htm, Retrieved July 1, 2009).
398
399
A
journal
of
the
build
and
natural
Environments,
No.
16
CSIRO Media release 98/213, Sept. 9, 1998. Retrieved July 23, 2009 from:
http://www.csiro.au/files/mediarelease/mr1998/WattleSeedAnotherIngredient.htm.
400
Di Cesare, L.F., Forni, E., Viscardi, D., Nani R.C. (2003) Changes in the chemical composition of
401
basil caused by different drying procedures. Journal of Agriculture and Food Chemistry, 51:
402
3575-3581
403
Djeridane, A., Yousfi, M., Nadjemi, B., Boutassouna, D., Stocker, P., Vidal, N. (2006) Antioxidant
404
activity of some Algerian medicinal plants extracts containing phenolic compounds. Food
405
Chemistry, 97: 654-660.
406
Drager, V.A., Garland, S.M., Menary, R.C. (1998) Investigation of the vatiation in chemicals
407
composition of Tasmannia lanceolata solvent extract. Journal of Agriculture and Food
408
Chemistry, 46: 3210-3213.
409
Haridas, V., Arntzen, C.J., Gutterman, J.U. (2001) Avicins, a family of triterpenoid saponins from
410
Acacia victoriae (Bentham), inhibit activation of nuclear factor-kappaB by inhibiting both its
411
nuclear localization and ability to bind DNA. Proceedings of the National Academy of
412
Sciences, USA, 98(20): 10986-10988.
413
414
Hodgson, J.M.; Wahlqvist, M.L. (1992) Koori nutrition and health: a Victorian review. National
Better Health Program: Australia 1992.
415
Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J.A., Deemer, E.K. (2002) Development and
416
validation of Oxygen Radical Absorbance Capacity Assay for Lipophilic Antioxidants Using
417
Randomly Methylated β-Cyclodextrin as the solubility Enhancer, Journal of Agriculture and
418
Food Chemistry 50: 1815-1821.
419
420
Javanmardi, J., Stushnoff, C., Locke, E., Vivanco, J.M. (2003) Antioxidant activity and total
phenolics content of Iranian Ocimum accessions. Food Chemistry, 83: 547-550.
17
421
Kahkonen, M.P., Hopia, A.I., Vuorela, H.J., Rahua, J-P., Pihlaja, K., Kujala, T.S., Heinonen, M.
422
(1999) Antioxidant activity of plant extracts containing phenolic compounds. Journal of
423
Agriculture and Food Chemistry, 47: 3954-3962.
424
425
Liu, R.H. (2004) Potential synergy of phytochemicals in cancer prevention: mechanism of action.
Journal of Nutrition, 134 (12S): 3479S-3485S.
426
Liu, H., Qiu, N., Ding, H., Yao, R. (2008) Polyphenols content and antioxidant capacity of 68
427
Chinese herbals suitable for medicinal and food uses. Food Research International, 41: 363-
428
370.
429
430
431
432
433
434
Madsen, H.L., Bertelsen, G. (1995) Spices as antioxidants. Trends in Food Science and
Technology, 6: 271-277.
Maiden, J.H. (1889) The useful native plants of Australia (including Tasmania). Turner and
Henderson, Sydney (1889) Facsimile ed. Compendium Pty Ltd., 1975.
Miller, J.B., James, K.W., Maggiore, P.M. (1993) Tables of composition of Australian Aboriginal
foods, Aboriginal Studies Press, pp. 256.
435
Ou, B. Hampsch-Woodill, M., Prior, R. (2001) Development and validation of an improved
436
oxygen radical absorbance capacity assay using fluorescin as the fluorescent. Journal of
437
Agriculture and Food Chemistry, 49: 4619-4626.
438
Ossipov, V., Nurmi, K., Loponen, J., Haukioja, E., Philaja, K. (1996) HPLC separation and
439
identification of phenolic compounds from leaves of Betula pubsecens and Betula pendula.
440
Journal of Chromatography A, 721: 59-68.
441
Prior RL, Wu X, Schaich, K. (2005) Standardized methods for the determination of antioxidant
442
capacity and phenolics in foods and dietary supplements. Journal of Agriculture and Food
443
Chemistry, 53: 4290-4303.
444
445
446
447
Rice-Evans, C., Miller, N.J., Paganga, G. (1996) Structure-antioxidant activity relationship of
flavonoids and phenolic acids. Free Radicals in Biology and Medicine, 20(7): 933-956.
Ruberto, G., Baratta, M.T. (2000) Antioxidant activity of selected essential oil components in two
lipid model systems, Food Chemistry, 69: 167–174.
448
Singleton, V.L., Rossi J.A. (1965) Colorimetry of total phenolics with phosphor- molybdic-
449
phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16: 144-158.
450
Sloan, A.E. (1005) Top 10 global food trends. Food Technology, 59: 20-32.
18
451
Southwell, I. (1996) Lemon myrtle – the essential oil. Australian Rainforest Bushfood Industry
452
Association Newsletter, 1: 6-7.
453
(http://www.cse.csiro.au/research/nativefoods/crops/myrtle.htm; Retrieved July 17, 2009)
454
Southwell, I., Russel, M., Birmingham, E., Brophy, J. (1996) Aniseed myrtle – leaf quality.
455
456
457
Australian Rainforest Bushfood Industry Association Newsletter, 4: 13-15.
Wojdylo, A., Oszmianski, J., Czemerys, R. (2007) Antioxidant activity of phenolic compounds in
32 selected herbs. Journal of Agriculture and Food Chemistry,105: 940-949.
458
Vazquez-Oderiz, M.L.; Vazquez-Blanco, M.E.; Lopez-Hernandez, J.; Simal-Lozano, J.; Romero-
459
Rodriguez, M.A. (1994) Simultaneous determination of organic acids and vitamin C in green
460
beans by liquid chromatography. Journal of AOAC international, 77: 1056-1059.
461
462
Zheng, W., and Wang, S.Y. (2001) Antioxidant activity and phenolic compounds in selected herbs.
Journal of Agriculture and Food Chemistry, 49: 5165-5170.
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483
484
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400
UV-320 nm
TP
1
200
2
496
497
498
80
UV-370 nm
TP
3
1
40
4
2
499
500
6
UV-520 nm
TP
150
100
5
50
501
502
503
10
20
30
40
50
Retention time (min)
504
505
506
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Figure 1. HPLC profiles of a crude extract from the Tasmannia pepper berry: 1 –
chlorogenic acid, 2 – coumaric acid, 3 – rutin, 4 – quercetin, 5 – cyanidin 3glucoside, 6 – cyanidin 3-rutinoside.
508
509
510
511
512
513
514
515
516
517
20
R1 = glucose; Cyanidin 3-glucoside
R2 = rutinose; Cyanidin 3-rutinoside
518
519
520
521
522
523
524
525
526
Chlorogenic acid
Rutin
Caffeic acid
Quercetin
Figure 2. Molecular structures of phenolic compounds detected in the Tasmannia
pepper berry.
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
21
544
Table 1. Total phenolics, vitamin C content and antioxidant capacity of selected native Australian herbs and spices.
Sample
Tasmannia pepper berry
Tasmannia pepper leaf
Anise myrtle
Lemon myrtle
Bush tomato
Wattle seed
556
557
558
559
560
561
562
563
Total phenolic
content
(mg GA Eq/g
DW)
16.9 ± 0.7*
102.1 ± 1.23
55.9 ± 4.7**
31.4 ± 5.9
12.4 ± 0.9
0.8 ± 0.12
Vitamin C
(mg /gDW)
ND
ND
0.7 ± 0.1
ND
ND
ND
Total Reducing
Capacity (FRAP)
(µmol Fe+2/g DW)
ORAC-H
(µmol
TEq/gDW)
ORAC-L
(µmol
TEq/gDW)
332. 9 ± 19. 9
779.5 ± 82.6
1314.5 ± 67.9
2158.0 ± 88.5
1225.3 ± 72.2
206.2 ± 9.0
17.8 ± 1.2
3504.4 ± 392.5
2446.1 ± 242.1
1889.8 ± 206.6
912.8 ± 117.7
53.4 ± 7.9
176.9 ± 2.7
572.7 ± 27.6
119.7 ± 0.1
1470.1 ± 171.9
18.6 ± 2.2
8.1 ± 0.4
545
ORAC-T
546
(µmol
547
TEq/gDW)
548
549
956.4
550
4077.1
551
2565.8
552
3359.9
553
931.3
61.5554
555
*Values represent means ± SD, n=3; **Total phenolic content is corrected for ascorbic acid; ND: not detected;
mg GA Eq/g DW: mg gallic acid equivalents/g dry weight; FRAP: Ferric Reducing Antioxidant Power; ORAC-H: Oxygen Radical
Absorbance Capacity-hydrophilic compounds; ORAC-L: Oxygen Radical Absorbance Capacity-lipophilic compounds; ORAC-T : total
ORAC activity; µmol TEq/gDW: micromole trolox equivalent/g dry weight.
564
565
566
567
568
569
22
570
Table 2. Mass spectrometric details and concentration of phenolic compounds in six native Australian herbs and spices (mg/gDW).
MS/MS
571
572
Tasmannia pepper
Berry
Leaf
Anise
Myrtle
Lemon
Myrtle
Bush
Tomato
Wattle
Seed
-/-/-
ND
ND
ND
ND
ND
ND
ND
ND
0.3±0.1
0.8±0.1
ND
ND
-/353
-/179
-/163
-/191
-/135
-/119
1.5±0.1
1.1±0.1
ND
30.0±0.2
ND
15.3±0.5
7.8±0.1
ND
ND
ND
ND
ND
0.4±0.1
T
T
T
ND
ND
Quercetin
Quercetin hexoside
Quercetin pentoside
Rutin
Rutin hexoside
Kaempferol/luteolin hexoside
303/465/449/611/609
773/449/447
153/303/303/303/301
611, 303/287/285
0.9±0.1
ND
ND
2.1±0.2
ND
T
17.9±0.3
ND
ND
T
ND
ND
ND
T
3.4±0.1
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
T
ND
T
ND
T
ND
T
ND
ND
T
T
Myricetin
319/317
153/151
ND
ND
4.1±0.1
3.6±0.1
ND
ND
Hesperetin rhamnoside
Hesperetin pentoside
Hesperetin hexoside
450/435/465/
303/303/303/-
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.8±0.1
T
4.2±0.1
ND
ND
ND
ND
ND
ND
Cyanidin 3-glucoside
Cyanidin 3-rutinoside
449/595/-
287/287/-
23.9±1.4
55.3±2.7
1.3±0.1
ND
ND
ND
ND
ND
ND
ND
ND
ND
Compound
[M+1]+/[M-1]-
Fragments
(m/z) (+/-)
Hydroxybenzoic acid
Ferulic acid
-/137
-/193
Chlorogenic acid
Caffeic acid
p-Coumaric acid
T= trace, ND = not detected
23