Medicinal herbs

- A preliminary evaluation of Astragalus, Bilberry, Feverfew and Stevia

A report for the Rural Industries Research and Development Corporation

by Rowland Laurence

May 2006

RIRDC Publication No 05/185 RIRDC Project No UT-34A

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© 2006 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 248 4 ISSN 1440-6845 Medicinal herbs –A preliminary evaluation of Astragalus, Bilberry, Feverfew and Stevia Publication No. 05/185 Project No. UT-34A The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details Dr Rowland Laurence Tasmanian Institute of Agricultural Research PO Box 3523 Burnie Tasmania 7320 Phone: 03 6430 4901 Fax: 03 6430 4959 Email: Rowland.Laurence@utas.edu.au

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4819 Fax: 02 6272 5877 Email: rirdc@rirdc.gov.au. Website: http://www.rirdc.gov.au Published in May 2006

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Foreword The increasing profile of traditional medical treatments in recent years has increased the demand for the quantity and the quality of medicinal herbal products, through both improved purity and reliability with regard to the dosage rates of the active ingredients contained. Pharmacological studies and clinical trials continue to find scientific support for the traditional claims made for herbal remedies and this improvement in the understanding of their effects has led to increasing sophistication and segmentation in their markets. Indeed, the delineation of the markets for herbal remedies and conventional pharmaceuticals has become increasingly blurred, with manufacturers of the latter taking a larger stake in the future of developments in herbal remedies. These market changes offer opportunities to Australian companies, whose operations encompass both field production and post-farm processing, in terms of both quality assurance and cost-efficiency, but these opportunities may be tempered by the long lead times needed to overcome traditional market loyalties and the amount of research and development required to bring to market the products of any one plant species, for which the individual market may not be large. This report reflects the introduction and preliminary observations on four such plant species, believed to have potential for the production of herbal extracts in Australia. This project was funded in major part from RIRDC Core Funds, which are provided by the Australian Federal Government. The project was also funded by financial and in-kind contribution from the company Botanical Resources Australia and in-kind support from the Tasmanian Institute of Agricultural Research. This report, a new addition to RIRDC’s diverse range of over 1500 research publications, forms part of its Emerging New Industries (Essential Oils and Plant Extracts) R&D program, which aims to support the growth of a profitable and sustainable essential oils and natural plant extracts industry in Australia. Most of our publications are available for viewing, downloading or purchasing online through our website: downloads at www.rirdc.gov.au/reports/Index.htm purchases at www.rirdc.gov.au/eshop Peter O’Brien Managing Director Rural Industries Research and Development Corporation

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Contents Foreword .........................................................................................................................................iii Executive Summary ......................................................................................................................... v 1. Introduction .................................................................................................................................. 1

Background .................................................................................................................................. 1 Literature review .......................................................................................................................... 2 Industry and research collaboration ............................................................................................. 6

2. Objectives and approach of the project........................................................................................ 7 Objectives..................................................................................................................................... 7 Approach ...................................................................................................................................... 7

3. Market trends ............................................................................................................................... 8 4. Astragalus investigations .......................................................................................................... 11

Acquisition of plant material...................................................................................................... 11 Extraction and analyses.............................................................................................................. 11 Field plantings............................................................................................................................ 12

5. Bilberry investigations .............................................................................................................. 20 Acquisition of plant material...................................................................................................... 20 Early growth............................................................................................................................... 20 Propagation ................................................................................................................................ 20 Field plantings............................................................................................................................ 20 Temperature and shading investigations.................................................................................... 23 Overseas study ........................................................................................................................... 24

6. Feverfew investigations ............................................................................................................ 26 Variety comparison .................................................................................................................... 26 Seasonal growth analysis ........................................................................................................... 27

7. Stevia investigations.................................................................................................................. 30 Acquisition of lines .................................................................................................................... 30 Glasshouse propagation ............................................................................................................. 30 Seed collection and germination ................................................................................................ 31 Initial field planting, March 2002 .............................................................................................. 31 Field trial planting, November 2002 .......................................................................................... 31 Expanded field plantings, 2003-04 ............................................................................................ 32

8. Conclusions ............................................................................................................................... 42 9. Bibliography.............................................................................................................................. 43 Appendix 1 ................................................................................................................................... 491 Appendix 2 ..................................................................................................................................... 61 Acknowledgements ........................................................................................................................ 89 Abbreviations ................................................................................................................................. 89 About the author............................................................................................................................. 90

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Executive Summary This report describes preliminary investigations into the production of four medicinal herbs in Tasmania. The work reflects the increased global market demand for medicinal herbs in recent years and the interest of the industry stakeholder company in this project, Botanical Resources Australia, in the opportunity to supply plant material and extracts from Australia. A proposal for the preliminary investigation of ten extractive herb species was considered by RIRDC in 2000, after which, the number of species to be investigated was reduced to four, these being evening primrose, astralagus, bilberry and stevia. Later discussions with an overseas consultant, regarding the poor marketing prospects for evening primrose at that time, led to a decision to discontinue evening primrose investigations and to include feverfew in the project work. The four species, astragalus, bilberry, feverfew and stevia are thus the subject of this report. The work has attempted to supply information on the suitability of these species for commercial production in south-eastern Australia by providing evidence of their ability to grow in a local, temperate climate, together with an indication of their commercial potential through plant and extract yield. Project activities involved acquisition of genotypes, plant propagation, seasonal analyses of growth and analyses of extracts, where available. In addition, the project sought to supply additional market information to that available, by seeking information on market trends in Europe. The roots of several astragalus species, but principally Astragalus membranaceus (Fisch.) Bunge and A. mongholicus Bunge (Radix astragali, huang-ch’i in China and hwanggi inKorea), are used in traditional Chinese medicine primarily as a tonic for the treatment of respiratory tract infections and against the common cold. However, therapeutic applications also exist in immune system enhancement and treatment of cardiovascular disease, through anti-hypertensive and vaso-dilatory effects, several of which are supported by clinical pharmacological data. These pharmacological effects are associated with several polysaccharides and saponins, principally, the triterpene saponins named astragalosides I-VII and iso-astragalosides I and II. Antioxidant activity has also been reported. Several species, particularly, A. gummifer, produce an edible gum exudate, gum tragacanth, which is one of the few permitted for pharmaceutical and food use. In the current work, analyses were restricted to Astragalosides I (the accepted reference standard), II and IV. Astragalus membranaceus was easily established in the field and grew well. Field plantings also wintered well after a first season but less well after a second, and its persistence may require further study if aged roots are preferred in Asian markets. Yield of dried roots from a fifteen month-old crop was calculated to be 1328 kg per hectare. Levels of astragalosides determined in the current study were high compared to published data, with a mean value for Astragaloside I over three sites being 0.25 per cent of dried roots. Data presented here also suggest that levels of astragalosides may increase with plant age, which may increase the attractiveness of further agronomic study to maximize extract yields. Feverfew (Tanacetum parthenium (L.) Schulz-Bip. (previously Pyrethrum parthenium and Chrysanthemum parthenium), a member of the plant family Asteraceae, is a perennial herb used as an ornamental plant as well as for its medicinal value. The major active ingredients are sesquiterpene lactones, of which parthenolide is in major proportion and preparations are used to prevent migraine headaches, fever and arthritis. Parthenolide is present in leaves, flowering tops and seed but levels vary greatly with genotype and morphological characteristics, and with seasonal growth. The degradation of the active ingredient in storage and the wide range of concentrations present in commercial preparations may explain why the product’s effects are sometimes questioned. The preliminary evaluation of feverfew carried out in this project has found that the species grows and winters very well in the local temperate climate. Parthenolide yields have been found to vary considerably with genotype, season and plant management. Parthenolide contents in the top-regrowth (approx. 200mm) after trimming plants of five genotypes varied between 3.6 and 25.5 mg per kg, providing calculated parthenolide yields of between 4.9 and 32.1 kg per hectare. Parthenolide contents of

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dried top-growth of un-trimmed plants of one genotype reduced from 7.1 to 2.9 mg per kg between October and February. While the market for feverfew at the time of writing this report is well supplied, these encouraging results have convinced the industry stakeholder, BRA, to maintain feverfew plantings beyond the life of the current project. Bilberry, Vaccinium myrtillus L., is one of some 150 species of Vaccinium, some of which, such as blueberry and cranberry, are widely cultivated for their berries. Bilberry is usually wild-harvested from cool-temperate forest under-storey at the present time. Fruit extracts of bilberry and other Vaccinium species are rich in flavonoids and their extracts exhibit high antioxidant activity which, in turn, can provide inhibition of some enzymatic effects of tumour promoting compounds. Levels and the range of such antioxidants in bilberry are about three times those found in blackcurrant. Vitamins, particularly A and C, are also present. After a slow period of establishment for bilberry, field plantings in Tasmania have flowered and produced a few fruit in their third year of growth, which timing is consistent with its growth in native habitats. Comparative measurements at two field sites have shown that plant growth at a high altitude site in Tasmania was more than 50 per cent greater over a growing season than that at a coastal, milder location. Additionally, when flowering commenced in the third year of the plants’ growth, flower numbers were also more than 50 per cent greater at the high altitude site. Insufficient fruit was obtained in this work to investigate extracts and further study will be needed to determine the yield potential of harvested fruit and extract in the Tasmanian environment. Indeed, the growth and yield responses of bilberry to seasonal temperature and shading regimes need to be better determined. Solutions to likely problems in mechanical fruit harvesting also remain and any development will require input over the longer term. Stevia rebaudiana Bertoni is native to Paraguay but has a wide climatological range, having been grown successfully from tropical to cool temperate locations. The leaves contain several natural sweeteners, diterpene glycosides, named steviosides and rebaudiosides. These glycosides are heat-stable and are not digested and thence have no calorific value as food, which attribute represents a strong market driver for stevia products in developed countries, where soft drinks with high calorific value are considered to be one factor in increasing levels of obesity and related disease. The product is traded globally, with Japan and some South American countries the major markets. Stevia grew well in the North-west Tasmanian summer but some plant loss occurred over winter months, suggesting that annual sowings may be preferable. Yields of plant dry matter, of which about fifty per cent was extractable leaf, were calculated to be 4834 kg per ha at one coastal site and 6317 kg per ha at an inland site experiencing high summer temperatures. Levels of stevioside and rebaudioside were generally consistent with published levels, particularly as some genotypes assessed were unimproved. Across ten genotypes, levels of stevioside varied between 10.2 and 2.9 per cent of leaf dry weight, and levels of rebaudioside varied between 10.6 and 0.6 per cent of leaf dry weight, with the influence also of site and season.

A current detraction to the development of stevia is the banning of the importation of stevia products by the European Union, its restriction in the USA and the possibility of its review in Japan, a major market, due to questions of safe use. While some food products containing stevioside as a dietary supplement have been marketed in Australia, imports of stevia products into Australia have recently been restricted. For these reasons, BRA has decided not to pursue the development of this species at this time.

Steady growth of markets in Europe is predicted but significant structural change is likely within the constituent countries of the expanding European Union, which will be driven by new EU directives being prepared. These directives attempt to bring tighter controls to the herbal medicine and supplement markets, to give a positive assurance of safety and enhance free trade. These issues are discussed and a commissioned report on the European market situation is provided.

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1. Introduction Background Herbal medicine has been the premier form of healthcare throughout history and the World Health Organisation estimates that some eighty per cent of the world’s present day population still relies on herbal preparations for some part of its medical treatment. Many modern pharmaceuticals are prepared from plants or their synthetic equivalents and most of those still so derived are used for purposes reflecting their traditional use (Wilkinson et al., 2002). The potential to expand the medicinal herbs and pharmaceutical extracts industries in Australia has been recognized for several years. From an Australian industry perspective, an RIRDC Report of a workshop on R&D opportunities in medicinal herbs and pharmaceutical plant extracts, held in 1997 (Anon., 1997), overviewed the then current industry situation. It defined the following major objectives for Australian R&D programs: • Determine which medicinal herbs are most appropriate for production in Australia • Improve yield and quality of active constituents • Provide cost effective production systems • Achieve high quality Australian products with national and international acceptance

More specific strategies related to genotype development, weed control in organic systems, improving post-harvest handling and value-adding through improving the specification of constituents’ profiles, analytical methods and purity levels. The preliminary study of the commercial potential of four medicinal herb species in Australia reported here was precipitated by the interest shown by this project’s industry stakeholder in the results of an earlier marketing study. This study, carried out by Chicago-based consultants, Keane International Marketing Inc. (Anon., 1999), investigated the market potential for a range of medicinal herbs in the United States. It provided a consideration of the future market for a wide range of natural plant extracts and was funded by the RIRDC and Botanical Resources Australia (BRA).

BRA is an Australian company involved in the contract growing, processing and export of refined products of natural pyrethrum. BRA is the second largest producer of pyrethrum products in the world and this success of its pyrethrum industry is based upon a strong industry focus on R&D, the collaborative support of local, experienced growers, a climatic environment conducive to excellent growth and yields and value-adding capability, such as accredited laboratories, a solvent extraction plant and a CO2-based refinery. BRA is keen to diversify into other opportunities and believes that these strengths, linkages and processing can be used, in the current expanding global market for medicinal herbs and nutraceutical products, to support a new Australian industry. The consultants’ report referred to above noted the increasing globalization of the industry and the increasing level of consolidation within it. Multinational chemical companies were seen to be taking an increasing interest and stake in the nutraceutical industry, which was predicted to expand in developed economies at a rate exceeding 10 per cent per year. These predictions, made in 1999, have largely come to pass and the market has seen increased diversification and linkage to markets for supplements and so-called functional foods. The Keane Report was emphatic with regard to the market’s directions towards standardised extracts and the role of improved extraction procedures, verifiable characterisation and quality/purity protocols. These issues are discussed further in this report. The report concluded that, while medicinal herb crops are a major growth area globally, there are good opportunities in the USA for Australian production of some of these products.

About half of the three hundred or so natural products in the flavour and fragrance industries are from cultivated plants and often overlap the flavour/fragrance and essential oil/natural extract/medicinal segment boundaries. In its recommendations, the report focused on products which had potential to fill a current demand and recognised the strengths and weaknesses of both Australia’s and BRA’s position in

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expanding in the global marketplace. The report considered twenty-two crops as worthy of consideration in Australia. Some of these, such as boronia, echinacea and garlic, are already under commercial development. A proposal for the preliminary investigation of ten extractive herb species was considered by RIRDC in 2000. After review of the proposal, the number of species to be investigated was reduced to four, these being evening primrose, astragalus, bilberry and stevia. After discussions with a consultant in England in July, 2001, regarding the poor marketing prospects for evening primrose, it was agreed between all stakeholders that the investigation of this species should be discontinued, and feverfew was included in the project’s work later in 2001. The four species, feverfew, astragalus, bilberry and stevia are thus the subject of this report.

Literature review The literature pertaining to herbal medicines is vast, stretching from ancient texts, traditional herbals, pharmacopoeias, topical books and journals and scientific publications, themselves extending from botany, plant biochemistry, pharmacy, clinical pharmacology and medicine. This review is restricted to the description, distribution, the extractives and uses of the four subject species, the changing marketplace for these herbs and to an Australian industry’s role therein. Astragalus membranaceus

Radix Astragali is the dried root of Astragalus membranaceus (Fisch.) Bunge and/or A. mongholicus Bunge, also known as milk vetch (US), huang-ch’i (China), ogi (Japan) and hwanggi (Korea), which are native to northern China and Mongolia, where they are associated with pine trees and sandy soils. Both species are perennial herbs and up to around one metre in height. They are now widely distributed in China, Siberia and northern Korea, where they are cultivated for medicinal purposes. The roots are cylindrical, fibrous and wrinkled, often over 50 cm in length but only around two cm in diameter. Morphological and chemical differences between the cultivated species have

been described (Lin et al., 2000; Cheng, 2000). The genus Astragalus (Tourn.), belonging to the family Fabaceae, contains some 1,600 species, many cosmopolitan. About 20 species, mostly those of Asiatic origin and particularly, A. gummifer, produce an edible gum exudate, gum tragacanth, which is one of the few permitted for pharmaceutical and food use by international regulators. The qualities of the gums (polysaccharides) vary between species and between plant parts. Uses are found in medicine, and in the textile, adhesives and beverage industries. In traditional Chinese medicine, Radix Astragali is used primarily as a tonic for the treatment of respiratory tract infections and against the common cold. Current therapeutic applications are in immune system enhancement and treatment of cardiovascular disease, through anti-hypertensive and vasodilatory effects. These uses are supported by clinical pharmacological data (Chang and But, 1987) and experimental pharmacology, both in vitro and in vivo with a range of animal species, and are well reported (Chu et al.,1988; Bombardelli and Pozzi, 1991; Zhou et al., 1990). Antioxidant activity has also been reported ( Toda and Shirataki, 1999). The extract majorly contains polysaccharides (astragalan, astraglucan), which are associated with immuno-stimulant activity and saponins, principally the triterpene saponins named astragalosides I-VII

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and iso-astragalosides I and II. Immune system enhancement has been shown through T-cell function assay, measures of increased interleukin-2 activity (Chu et al., 1988) and increases in phagocytic indices (Chang and But, 1987). Astragaloside I is a normal reference standard (Ganzera et al., 2001). Dosage normally occurs with the crude plant material at rates between 9-30 g/day or with extracts (Anon.2000). Vaccinium myrtillus

Vaccinium myrtillus L.is one of some 150 species of the genus Vaccinium belonging to the family Ericaceae. However, the taxonomy of the genus has been the subject of much debate, with hybridization and polyploidy occurring (Camp, 1942). Most species occur in the cool-temperate latitudes of Europe and North America, but some are also found in the Andes and Mongolia (Van der Kloet, 1988). Other species also cultivated or wild-harvested for their berries are the blueberry, V. corymbosum and V. angustifolium, the cowberry, V. vitis-idaea (sometimes called cranberry in error) and the true cranberry, V. oxycoccus. Bilberry is frequently co-dominant, with shrubs such as juniper and alder, in the understorey of coniferous and deciduous forests. It prefers semi-shade, although its tolerance of, and ability to set fruit in, full sunlight appears to depend on altitude and aspect (Glowacki, pers.comm., 2003). In Poland the yield of extract and actives, particularly vitamin C, has been shown also to vary with altitude (Glowacki, pers.comm., 2003). In lowland areas of Poland, propagation and fruiting only progresses well in shade of 40-50 per cent but at higher altitudes plants will grow and fruit in full sun. Above 500 metres altitude the species does not fruit in shade and fruit size is improved (Weglarz, pers. comm., 2003). Given the plant’s forest habitat, most published yields and yield models reflect ecological partitioning in total forest production rather than monoculture yield (Ihalainen and Pukkala, 2001). The plant is a small shrub, seldom taller than 40 cm, with a shallow, fibrous and matted, trailing root system. The stems bear alternate, simple, ovate leaves, which can turn red-brown in autumn or in full sunlight (Vanninen et al., 1988). Flowers are borne singly on new stems and are pollinated by bees. The fruits are globose berries, up to 8mm in diameter, which are usually purple to black, but occasionally red, in colour. They are an important food source for many animals, from heath-land birds to bears (Rogers, 1976). While seed is usually viable and germination shown to be promoted by several temperature treatments (Van der Kloet, 1983), seedling growth is extremely slow (Ritchie, 1956). Like many species of Vaccinium, bilberries require acidic soils and are commonly associated with raw humus and the large leaf litter loadings of the forest floor. Fruit extracts of bilberry, Vaccinium myrtillus, and other Vaccinium species are rich in flavonoids (proanthocyanidins and anthocyanins, Hakkinen et al., 1999) and exhibit high antioxidant activity (oxygen radical absorbance capacity, Pietta et al. 1998), which in turn provides an ability to inhibit some enzymatic effects of tumour promoting compounds (Madhavi et al., 1998). Nyman and Kumpulainen (2001) found the total anthocyanidin content of bilberry to be 360 mg/100 g compared with135 mg/100 g in black currant and 26 mg/100 mL in Cabernet Sauvignon red wine. Dugo et al. (2001) found 14 anthocyanin compounds in bilberry extracts compared with five in mulberry. Various vitamins, particularly A and C, tannins and antibiotics are also present. Extracts are said to assist in the strengthening of vascular capillaries and reducing varicose veins and arthritis. In addition, claims have been made regarding the ability of bilberry extracts to improve night vision. There is a small trade in the leaves of bilberry as an anti-diabetic remedy based on arbutin and other phenolics. In addition to fresh sales and medicinal preparations, fruit extracts sometimes are alleged to be used to colour white wine for sale as red wine, as the components are comparable.

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Tanacetum parthenium

Feverfew is a perennial herb and a member of the plant family Asteraceae. Several members of the genus Tanacetum (L.) may be collectively known as tansy, although this name is also more specifically used for T. vulgare, a plant with some traditional use as a flavouring but which can also be poisonous to mammals in quantity. Chysanthemum parthenium is a frequent synonym for feverfew. T. parthenium is native to south-eastern Europe but is now found around the world, being used as an ornamental plant as well as for its medicinal value, with the latter use first recorded by the ancient Greeks (Baranov, 1999). Several sesquiterpene lactones are the major active ingredients, of which parthenolide is in major

proportion and preparations are used to prevent migraine headaches, fever and arthritis. These active constituents are present in leaves, flowering tops and seed (Heptinstall et al., 1992) but parthenolide levels vary greatly with genotypic origin, morphological characteristics (Cutlan et al., 2000) and with seasonal growth (Hendriks et al 1997). The active ingredient has been shown to be effective against migraine headaches (Johnson et al., 1985; Vogler, 1998) but the whole, dried leaf has been shown to be more effective than extracts of feverfew (Heptinstall et al 1992). It also has been shown to degrade over time in storage (Mittra et al 2000). These findings, in addition to evidence that a wide range of concentrations of parthenolide are present in commercial preparations (Nelson, 2002), provide some explanation for the continuing debate regarding the products’ effectiveness. Extracts of the herb feverfew inhibit human blood platelet aggregation and secretion induced by a number of agents in vitro and this may relate to the beneficial effects of feverfew in migraine. (Groenewegen and Heptinstall, 1990). There is also evidence of anti-inflammatory activity, probably related to the presence of flavonoid glycosides, and the pharmacological basis for this has been investigated (Piela-Smith and Liu 2001, Kang et al., 2001). Conversely perhaps, repeated contact with feverfew has been reported to result in delayed hypersensitivity and cross-reactions with other Asteraceae genera containing related sesquiterpene lactones have been noted (Hausen and Osmundsen, 1983). Mild sedative activity, possibly due to the presence of pinenes, has also been reported (Bohlman and Zdero, 1982). The plant and its extract have been used, historically, as insecticides and recent research indicates that anti-feedant activity exists against Spodoptera and Callosobrucus at levels similar to those found with neem extracts, while other constituents of the extract have nematocidal and phytotoxic activity (Datta and Saxena, 2001). Stevia rebaudiana Stevia rebaudiana Bertoni is a herbaceous, bushy plant, native to Paraguay and one of over one hundred species of Stevia in the American tropics and sub-tropics. The species naturally grows to around 80 cm in eastern Paraguay at lower altitudes and high rainfall, which produce seasonally humid conditions. It is common in poorly drained, acidic, sandy soils. However, the plant has a wide climatological range, having been grown successfully from tropical to cool temperate locations, where it is restricted to summer seasonal growth as an annual.

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Local South American populations have used the species for many years as a sweetener for traditional teas and other drinks due to the presence of several diterpene glycosides in the leaves. Owing to increased interest in natural and non-calorific sweeteners in developed nations, there now exists a world trade of some 3000 tonnes of extracts. The plant has also been used to treat diabetes, hypoglycemia, hypertension and as a bactericide. The plant and its active constituents have been actively researched for many years, particularly in South America and in Japan, where most of the product traded globally is consumed. Many reviews and books are now available and the reader is directed to the report by Rank (1999), as a precursor to a current RIRDC project to develop stevia in Australia, listing 175 references. A recent monograph (Kinghorn, 2002) also provides an excellent situation summary.

The two glycosides which are present in stevia leaves in a major proportion are stevioside, which usually varies between five and ten per cent (w/w) of dried leaves and rebaudioside A which is present to around four per cent (w/w). Other glycosides having sweetening properties are also present in lesser amounts. Flavonoid glycosides and some essential oils are also present. Rebaudioside A is relatively sweeter than stevioside and has better flavour characteristics. The dried leaf extract is generally described as being about 300 times sweeter than sugar. These glycosides are generally heat-stable and are not digested and hence have no calorific value as food.This attribute represents a strong market driver for stevia products in developed countries, where soft drinks with high calorific value are considered to be one factor in increasing levels of obesity and related diseases. Breeding programs over several years have improved the levels of the glycosides in stevia, with current values about twice those of unimproved genotypes (Huang and Guo, 1996). The proportion of rebaudioside A, the glycoside preferred due to its greater sweetness and lack of after-taste has been increased relative to stevioside.

The expansion of markets for stevia products has been hampered in recent years by arguments surrounding safe use, notwithstanding its traditional consumption and anti-obesity benefits. These arguments concern the related compound steviol, which is a precursor in the biosynthesis of stevioside, and the probability of its being enzymatically activated, in vivo, to form a mutagenic metabolite. Steviol, in the presence of activation enzymes, has been shown to be mutagenic in some in vitro tests but not others (Matsui et al., 19960). Stevioside has been shown to be degraded by gut flora to steviol in rats (Nakayama et al., 1986) and metabolized by the microflora of human faeces (Hutapea et al., 1997) but to be non-carcinogenic in rats (Toyoda et al. 1997). On the basis of the debatable evidence, products containing stevia as a food additive (as a sweetener, for ‘technical effect’) have been banned in the United States since 1995, although its use as a ‘dietary supplement’ or an ingredient thereof is permitted (Anon., 1996). All uses of stevia, except as an ornamental plant, have been banned by the European Union (Anon. 1999), although there remains active lobbying for the re-instatement of some uses. In Australia, applications have been lodged in the past with the Australia New Zealand Food Safety Authority for the use of stevioside as a food additive and one application was current at the time of the beginning of this investigation. Some food products containing stevioside as a dietary supplement have been marketed in Australia. However, imports of stevia products into Australia have recently been restricted (AQIS imported food notice 02/05 - The use of Stevia and Stevioside in food). The plant is usually grown from seed, which needs to be fresh and handled carefully in germination. Old seed quickly loses viability. In current commercial situations, plant populations around 8 per sq m are targeted. The whole plant (above ground) is harvested in the ‘flower-bud’ stage and sun or kiln dried. Leaves are stripped from stems and extracted with boiling water, which removes about 95 per cent of the glycosides. While few commercial yields have been reported, they are thought to be in the order of 1500-2500 kg dry leaf per hectare. In warm climates, the plant is easily ratooned, which can reduce production costs.

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Industry and research collaboration The work reported here was carried out in close association with the industry stakeholder, Botanical Resources Australia. This project represents an interest of this company to diversify into other opportunities and the company believes that its strengths in market linkages, production systems and extraction/processing, can be well used in the current expanding global market for such products, to support a new medicinal herb crop industry. In addition to its cash contribution to this project, the company was responsible for in-kind support in the form of laboratory analyses of plant material in its NATA accredited laboratory and some field activities, where crop husbandry required commercial equipment. Growers also collaborated with the lease of experimental field sites. This project’s work on stevia has been carried out in collaboration with Professor David Midmore and Andrew Rank of the University of Central Queensland, who have investigated the potential of stevia in tropical and sub-tropical Australia, also with the support of the RIRDC. Tasmanian data have been supplied to this project to extend the latitudinal range of results.

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2. Objectives and approach of the project

Objectives In determining the appropriateness of the four selected medicinal herb species for production in south-eastern Australia, the current project targeted the first of the objectives for the Australian industry defined during the RIRDC-funded workshop on R&D opportunitiesreferred to in Section 1.1 (cf. Anon., 1997). The work has attempted to provide information on the suitability of astragalus, bilberry, feverfew and stevia to commercial production in south-eastern Australia. The objectives were introductory in nature – to provide evidence of the ability of the nominated plant species to grow in this cool, temperate climate and, further, to provide indication of commercial potential for plant and extract yield. In addition, the project was to attempt to provide additional market information to that available from the earlier referred to Keane report (cf. Anon., 1999) and other local reviews, by supplying information on market trends in Europe. Approach The initial approach in this work was to seek and introduce a range of genotypes of each species into Tasmania, to propagate under greenhouse conditions and thence in small field plots for observation. Progress with such propagation proved to be slow, particularly with bilberry and stevia. In the case of of bilberry, its propagation was not well suited by ambient greenhouse conditions. In the case of stevia, poor germination and seedling losses were followed by significant numbers of plant fatalities in small field plots due to the seasonal conditions in the cool temperate climate, which proved to be severe on this species. This slow progress led to a review of approach in December 2002, after which a greater emphasis was placed on establishing fewer but larger field plots of the four plant species, in order to better address the need for plant and extract yield information. In chronological summary, the following greenhouse, field and laboratory activities were carried out as part of this project: In 2001-02, genotypes of the four species were sought and acquired. Establishment in a greenhouse was attempted and initial field plantings of feverfew and stevia were made. Where establishment from seed proved to be slow (bilberry) or difficult (stevia), methods of propagation were investigated. In 2002-03, field plantings of genotypes of the four species were completed, including larger-scale planting of Astragalus and plantings of some genotypes in sites deemed likely to be suited to individual species. Initial monitoring of plant growth was carried out. In 2003-04, monitoring and analyses of growth were expanded to include some comparison across sites. Stevia investigations included those on a genotype provided by University of Central Queenland collaborators. Analytical methods for the preparation of extracts were developed and the extracts analysed, with the exception of bilberry, some plants of which only commenced flowering in 2003-04 and provided occasional fruit set at one site in 2004-05. Overseas study by the author in July 2003 in Poland included observation of bilberry harvesting in Poland and related discussions with local scientists. Development of analytical methods for astragalosides in Astragalus proved to be slow and has resulted in time extension to complete the project. Additional information on market trends in Europe was sought during brief visits to Germany and England made by BRA representatives and the author.. During this visit contact was made with Austrade staff and, subsequently, Mr Robert Bergmann of Austrade Germany was contracted in January 2002 to provide market information on the four medicinal crop species of interest to this project. His report was received in July 2003. In July 2003 also, the author, in association with other work commitments, attended the 2003 Natural Products Expo Europe in Amsterdam, which provided information on proposed changes to herbal medicines legislation in Europe. These are reported below.

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3. Market trends The medicinal herb market in Australia is now worth about $60 million per year and continues to expand, having grown at about 15 per cent per year over the last few years. Globally, the majority of demand for medicinal herb products (about 75 per cent) lies in USA, Western Europe and Japan and collective market growth has also been in the order of 15 per cent per year in recent years. For example, sales of echinacea, a product traded in large quantity, have doubled since 1995. However, demand for specific products can be atypical of overall trends. For example, notwithstanding the market popularity of echinacea products at the time, discussions with the Hamburg-based company, Martin Bauer GmbH, in 2001 reflected little interest in the Australian product. Also, trade in St John’s Wort products increased by some 1900 per cent between 1995 and 2000 but then dramatically retracted due to publicity on safety issues. Kava products have similarly been withdrawn from sale in some countries due to health safety issues. Collectively, these issues have drawn responses from governments in the form of legislative programs for tighter product specification and improved pharmacological information of major product constituents. These changes, in turn, are impacting differentially on industry players. There are in excess of 800 companies marketing globally but many are recent and relatively small. Three-quarters of the global market is shared among fourteen well-established companies. Australia, with the exception of a few products, is a relatively new player in this market, which shows strong loyalties. However, new suppliers in Australia have an expanding local market with which to grow and local production infrastructure and the stability of the business environment are positive factors in sales of related products such as pharmaceutical poppy and pyrethrum extracts. Value-adding to products, through quality assurance guarantees, is necessary to meet future customer expectations and product safety legislation. In this changing market-place, Australian companies with integrated operations are well-positioned for future growth. Report commissioned from Austrade Germany The report commissioned from Austrade Germany (see Appendix 2) predicted the continuation of moderate growth in the European market, driven by consumer interest and government-led initiatives to encourage self-medication for a range of ‘minor’ conditions in order to relieve the financial burden on publicly-funded medical systems. Positive sales factors in Germany included an expansion of the “Negative List”, comprising those drugs not reimbursed by medical insurance schemes. However, the shift towards more generalized distribution and consequent growth in privately or ‘home-branded’ products is providing downward pressure on prices. A range of other factors, data and indicative predictions are provided in this report. Attendance at the 2003 Natural Products Expo Europe, Amsterdam The Natural Products Expo Europe was held on 3-5 June 2003 in the Rai Centre, Amsterdam. The Expo was owned and operated by Penton Europe, a division of Penton Media Inc., USA. As such, the media played a significant role in the event. With strong traditions in the use of medicinal products but with limited and often weak science in the support of their use and the use of functional foods, the claims made for efficacy were sometimes excessive. There were 154 exhibitors at the event, ranging among herbal remedies, functional foods and nutraceuticals, vitamins and mineral supplements and other ‘well-being’ products. Exhibitors of products relevant to medicinal plant raw materials, which are the subject of current TIAR research projects, were approached, in order to ascertain interest in these raw materials, which might be produced eventually in Tasmania. Those companies, which dealt in the relevant medicinal plant products and whose representatives showed interest in acquiring Tasmanian product were: • Juttner & Partner, Switzerland – dealt with bilberry leaf and fruit preparations. • Hasco-Lec Pharmaceutical Co., Poland – dealt in bilberry products. • Linnea SA, Switzerland and global links – purchased 50-100 tonnes of bilberry fruit per annum. Also

the contact specifically inquired about red clover hay, of which they required around 500 tonnes per

9

annum with an indicator price of EU 2.10 per kg. The contact also specifically asked after butterbur (Petasites hybridus) preparations.

• Nutralife UK Ltd – buys 50-100 tonnes per annum of Echinacea angustifolia from Brazil and has had trouble with consistent quality in the past. Is just beginning a research program to improve and grow echinacea itself. However there is a possibility of Tasmanian supply in the short to medium term.

• Pharmline Inc., USA – acquire most of a wide range of products from within the Americas but welcomed information on product availability. Products include astragalus, bilberry, echinacea and borage oil.

Information sessions were held continuously during the two-day period. While some were quasi-marketing presentations, the majority were panel discussions or presentations around market briefings. General comments coming from sessions There was general admission by several speakers that there is a lack of good science underpinning many claims concerning products in the market place. The term ‘hope marketing’ was used to describe the overall strategy of marketing the promise of a healthier life. Alternatively, many medicinal herb products have been used as relatively cheap medicines for many years and with the current costs of creating a new ‘conventional’ drug in the order of US$ 700-800 million, this demand is unlikely to diminish. There now seems to be evident a political awareness which, in response to the high costs of traditional care, drives public funding towards preventative systems, into which category medicinal herb products may be placed. There is, therefore, a need for a stronger scientific background on all medicinal herb products and a contributing need to get the support of the ‘conventional’ medical community. This would lead to science relating the remedial activity of a product to an understanding of the disease process in question and to its management through a treatment program, rather than just to product description and nebulous claims. However, most of such arguments came from those speakers who were medically qualified (in the ‘conventional’ sense) and were not embraced by all. Similar ‘conventional’ views were expressed in relation to the growing market for functional foods, where education of the general public in the area of basic human nutrition would be effective in improving healthy lifestyle situations and obviate many functional foods. For example, it was noted that fruit and vegetable intake is still declining world wide, despite the efforts of many governments, and the frequency of Type 2 diabetes is doubling every ten years. However, at present, the market is there and one felt that the audiences, generally, were not over-receptive to such conventional arguments! More interest was shown in the promotion, for example, of the Food and Health Marketing Handbook (www.thehandbook.com). New functional food streams are coming to the market continually, such as algal (Spirulina) preparations and grass/cereal (vegetative) extracts, which are becoming popular in the USA. A new European Foods Standards Agency has recently been introduced to assist in addressing the very different regulations, which currently exist across the EU (eg Vitamin A&D fortification of fats is common in UK but banned in Germany) and to attempt the difficult task of getting consistency between government regulations and industry standards. Those sessions, which focused on the European market situation, were almost totally consumed by the issues being presented to the industry by new EU (and thence member country) directives then being prepared. These directives were in various stages of final drafting before being published for comment later in 2004. They attempt to bring tighter controls to the herbal medicine and supplement markets. The UK and Dutch markets, which are currently very liberal in their regulation, will find the new regulations very difficult but the German, French, Spanish and Italian markets, which are now quite tightly regulated, will have less difficulty. The global aims of these directives are to give a positive assurance of safety and enhance free trade. In the UK, it is intended that directives will be completely in force in 2009. A Fortified Foods Directive will define ‘safe’ levels for vitamins and minerals in foods. A Food Supplements Directive will demand a safety dossier for ingredients towards registration of products. Currently, owners of some three hundred products intend to lodge dossiers. Such dossiers are required by June 2005. There will be a Products Directive for Foods for Intense Muscular Effort. A Traditional Herbal and Medicinal Products Directive

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was then being empowered. This will require scientific evidence to support claims concerning active ingredients. Products must be able to demonstrate 30 years of safe use and 15 years safe use in Europe. This is seen to restrict the innovative introduction of non-traditional plant extracts. Another criticism is that there appears to be no home for products which are mixtures of medicinals and supplements. Further information is available from the website of the Health Food Managers Association (www.hfma.co.uk). In the UK, a large-scale clinical trial of the efficacy of ginkgo preparations in the prevention and/or control of dementia is being undertaken. In Germany, prices and turnover of natural pharmaceuticals are currently low but functional foods, their ingredients and natural ingredients for cosmetics are booming. Germany is currently a strongly regulated market with pharmaceuticals, supplements and dietetics handled differently. A wide range of products is available. Eighty per cent of medical doctors in Germany prescribe herbal medicines and some are reimbursed by Government. This will change under the new directives but this issue of reimbursements is unresolved. It was argued that the new directives will leave a temporary lack of regulation of nutritional supplements and new regulations are likely to be tested in the courts. It was felt that small players and small turnover products would disappear from the German market under the new regulations. An update on the regulatory situation in Japan was also provided by a representative of the Japanese Health Food and Nutritional Food Association (JFNHA), an association supported by over 3000 manufacturers. Under what is referred to as the April 2001 framework, foods with health claims are divided into drugs (including quasi-drugs, some of which are considered foods in other countries), foods for specialised health uses (FOSHU), and foods with a nutrition function claim. These categories are specifically regulated. In addition, a wide range of ‘health foods’ is unregulated and these include those with a JHFA label (a standardised stamp of approval costing about EU 7800, including laboratory testing – currently acquired by some 840 products). Stevia rebaudiana extracts are currently in this unregulated section. There is no reference to functional foods in this structure and some food categories, such as foods for diabetics etc., are not dealt with under the regulated sections. FOSHU labeled products make claims to specific health benefits. Products are individually evaluated by Government and must have Japanese-based clinical data to support the application. Investment towards a FOSHU labeling may be around € 77,000. Recent approvals include oligosaccharides, lactobacillus preparations and soy products. Foods with a nutrition function claim, which are regulated, include vitamins, some minerals, α-linolenic acid and DHA. Maximum and minimum levels are specified and functional claims are specified. Japanese people currently consume more functional foods per head of population than any other country and water with added amino acids (‘functional water’) is a major seller at more than three times the price of bottled water. A discussion on business opportunities in traditional herbal medicinal products in Europe resulting from the new directions reflected upon the lack of product uniformity across Europe and the stagnation with respect to new products coming on to the market. The Traditional Herbal Medicinal Products Directive will eventually result in a positive listing of herbal ingredients and products. There are likely to be opportunities for products with specific combinations of ingredients. Factories will be licensed, products registered and specific attributes, such as shelf-life stability will be required. It was considered by the majority of speakers that the changes could be a major contributor to the development of what are currently stagnant markets. In markets where phytotherapy is currently growing, such as the UK, the directive should assist confidence and prevent the replication of some recent negative media ‘scares’. There was a reminder that the EU Directives will be interpreted by component national governments as best to provide for that nation’s situation. For example, in UK, the current ‘Medical Herbalists’ are to seek a state registration to continue operating without change. A final presentation on supply trends in the nutrition industry was provided by Nutrition Business Journal (www.nutritionbusiness.com), which predicted further shake-out in the US herbal supply and manufacturing industry, further targeting of demographics and further recognition that emotional rather than scientific benefits sell functional foods. For example, Pepsi is repositioning into more nutritional products and Nestle, going further, is repositioning into a company providing ‘well-being’ foods.

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4. Astragalus investigations Acquisition of plant material After contacting several research agencies and seed companies internationally between May and July 2001, three lines of Astragalus membranaceus seed were acquired. These were a line (Lot 515968) of South Korean origin from USDA ARS at Washington State University, the line S3991 from Richters, Canada and a line of unknown origin from B&T World Seeds, Olonza, France. These seed lines all exhibited reasonable germination and were sown into soil plugs in November 2001. Extraction and analyses Several methods for the determination of astragalosides were found in published literature, (e.g. Zhang ZiZhong et al., 2001) generally based upon Soxhlet extraction with methanol or methanol:water, followed by reverse-phase HPLC/photodiode array detection. However, many given Soxhlet extraction procedures were time-consuming and super-critical fluid extraction, using liquid carbon-dioxide as the primary solvent, was investigated as an alternative extraction technique, as this method has been used for extraction of grape glycosides, digoxin and saponins from anemone (Xiao-hong et al., 2003). In these investigations samples weighing 3 to 4 g were extracted at 30400 kPa and 50°C, with a static phase of 15 min, a dynamic phase of 90 min (CO2 flow rate of 1 g/min), 20% methanol modifier in the dynamic phase and a CO2 density of 0.88 g/ml. The collection solvent was 100% methanol. Different extractions were attempted without modifier in the static phase, with 20% methanol modifier in the static phase, with methanol spiking of the sample before extraction and with increased extraction temperature. While methanol additions to the static phase were beneficial, the initial results obtained from these super-critical fluid extractions were poor compared with Soxhlet extraction. With limited time available to complete the analyses Soxhlet extraction was re-employed, although further experimentation with super-critical fluid extraction may result in a faster and cheaper analytical technique. Soxhlet extraction was modified from that given in the Chinese Pharmacopoeia (Anon., 2000). Extraction using pure methanol and 80% methanol, replenishment of solvent and extraction vessels of varying size were compared, as were extraction times up to 3.5 hr. It was found that 80% and 100% methanolic extractions were similarly effective, that small extraction vessels were more effective in a given time than large extraction vessels, that solvent replenishment was not worthwhile and that a 2hr extraction time returned in excess of 90% of the astragalosides returned by a 3.5 hr extraction. A 2hr extraction time in small (100 ml) Soxhlet vessels was used, therefore, with 150 ml of 80% methanol as extractant. Analysis of extracts followed the method of Ma et al. (2002), with modifications after Ganzera et al. (2001). An LiChrospher C18, 150mm by 4.6mm column was employed, with water and acetonitrile mobile phases. Standards for Astragalosides 1, 2 and 4 were obtained from Chromadex, USA. The purity of these was reduced over the period of analytical method development with the appearance of breakdown products. As a result of this, absolute values of astragalosides determined in this study should be compared to published levels with caution, although comparisons within the present study are unaffected.

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Field plantings 2002-03 Methods A field site on krasnozem soil (pH 6.1, 82 ppm P and 511 K) was prepared at Forthside Research and Demonstration Station (FRDS), near Devonport, Tasmania (lat. 41° 12min S, long. 146° 16min E; elev. 130 m; ann. rainfall 1013 mm) to which 300 kg per ha of 7:12:9 fertiliser were applied. Weed matting was used to assist weed control and seedlings were planted, on 25 March 2002, into three-row beds, each 1.6 metres wide and 12 metres long, of which there were six replicates of each seed line. An appropriate inoculant was watered onto the plants after planting. Growth was monitored and plots maintained until March 2004. Whole root samples were taken from one plant in each of three plots of each genotype on 19 February 2003. Each root was washed, dried, ground in a laboratory hammer mill and provided to Botanical Resources Australia’s laboratories, where they were held at 4°C pending extraction and analysis. Given the successful growth and wintering of plants at this site, additional seed was acquired from B&T World Seeds, inoculated and sown, on 1 November 2002, directly into a single plot, 0.1 hectare in area, which had been prepared within a commercial paddock at Kindred, near Devonport. Growth of plants was again monitored for the following two seasons and plots maintained until March 2004. Whole plant samples were taken from four, randomly-selected areas of two sq metres each on 4 March 2003. Plants from each plot were collectively washed and plant fresh weight, and root fresh and dry weight noted. Root samples were ground in a laboratory hammer mill and provided to Botanical Resources Australia’s laboratories for extraction and analysis. An additional, mechanized sowing of inoculated B&T seed was also made on 23 January 2003 at another local, commercial paddock (Werrin Farm) operated by the project stakeholder, BRA. Two beds were sown, each 1.6 metres wide and 30 metres long. Growth was again monitored but no destructive analysis of plants was attempted in season 2002-03. Results and discussion At the first-planted site, FRDS, seedling plants grew well for the remainder of the season, notwithstanding the appearance of some fungal leaf spotting. A small taproot was produced by the time vegetative growth died back during May 2002. All plants were reduced to a root crown during winter conditions and regeneration of top growth commenced in October 2002. Re-growth of over 75 per cent of plants was noted in late November 2002. This trial site was exposed to strong westerly winds during autumn and winter. The best-regenerated plot populations were present in the more sheltered area of the trial. These regenerated plants began to flower in mid-January 2003. No significant difference was found between the three lines with respect to earliness of flowering, as measured by the number of plants in flower per plot on 29 January 2003, when 41 per cent of all plants were flowering. Flowering stems reached a height of around 150 cms. Seed was set freely and the (leguminous) seed-pods dehisced on maturity. The mean weights of dried roots, taken from three plants of each of the three genotypes on 19 February 2003 at FRDS, together with their astragaloside contents are shown in Table 1.

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Table 1. Mean weights of dried roots of individual astragalus plants of three genotypes, harvested on 19 February 2003 at FRDS, together with their astragaloside contents.

Genotype Root dry wt

(g) Astragaloside 1 (% dry wt)

Astragaloside 2 (% dry wt)

Astragaloside 4 (% dry wt)

Total A1, A2, A3 (% dry wt)

Richters 40.3 0.277 0.114 0.043 0.433

USA 61.0 0.323 0.127 0.036 0.485 B&T 48.4 0.248 0.111 0.035 0.393

Mean 49.9 0.283 0.117 0.038 0.437

Sig. (P=0.05) ns ns ns ns ns

There were no significant differences between the three genotypes in relation to any of these attributes. Levels of the three astragalosides measured were high (around double) compared with some published data. At the Kindred site, emergence was slow, with a mean population of 8.8 seedlings per sq m being achieved six weeks after mechanised seeding. This figure rose to 12.2 seedlings per sq m after a further eight weeks. While this population was lower than that targeted (30 plants per sq m), this area also grew well and flowered in late February 2003. The mean weight of dried roots taken from four, randomly-selected areas of two sq metres each of this B&T line on 4 March 2003 at Kindred were 315.8 g, 238.3 g, 266.4 g and 241.8 g respectively, indicating a mean dried root yield of 1328 kg per hectare for this fifteen month-old crop. At the Werrin Farms site, a mean population of 26.0 plants per sq metre was achieved two weeks after sowing and was near that targeted. Plants reached a mean height of 22.2 cm by end June 2003, without flowering, at which time winter die-back of tops had commenced. 2003-04 Methods In December 2003, the replicate plots of the three seed lines planted at FRDS in March 2002 were harvested and roots from each plot dried and weighed. An analysis of variance was carried out on these data (Table 2). Additionally in season 2003-4, whole plant samples (B&T Seeds line) were taken sequentially from plantings at the three sites, FRDS, Kindred and Werrin Farms, to investigate seasonal growth and changes in active root constituents. On six dates between mid-October 2003 and mid-March 2004, eight plants were randomly selected from each site. Plant height, primary stem number, taproot length and fresh and dry weights of top growth and roots were recorded for each plant. For the first five times of harvest only (October 2003 –February 2004 inclusive) at the three sites, the dried roots were ground individually and were sub-sampled for extraction and analysis by the method described above. Given that plants at these three sites were respectively 23 months, 11 months and nine months old at the start of this sequential harvesting in October 2003, it was considered that these data, although confounded by site differences, might provide some information on the effect of plant age on root and extract yield. These three field sites were of similar krasnozem soil-type, similar elevation and were within five kilometers of each other, although differences in aspect and slope were present. Site history and soil and rainfall data were also unavailable for the Kindred and Werrin Farms locations. Analysis of variance

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was carried out to determine whether significant differences in the measured attributes, including astragaloside levels, were present between these three sites over the five-month period, with the (eight) individual plant values used for replication. Results and discussion No significant difference between the dry root yields of the three lines in the FRDS trial was found (Table 2). The lower mean dry root yield of these two-year old plants compared with those above harvested at Kindred in March 2003 is consistent with a lower plant population at the Forthside site.

Table 2 Mean dry root yield (kg/ha) of two-year old plants of three Astragalus seed lines.

Mean dry root yield (kg/ha)

B&T 998 Richters 1276 USA 1210 Mean 1161

Sequential harvesting of plants at the three sites, FRDS, Kindred and Werrin Farms found significant differences in measured parameters with respect to site and harvest date. Stem numbers increased during the season at all three sites but more stems were produced by (older) plants at FRDS than by those plants at other sites (Table 3) and the three mean site values were directly correlated with plant age. Table 3. Mean number of stems per plant of Astragalus at three sites and six times in 2003-04.

Harvest date FRDS Kindred Werrin Farms Mean 9-Oct-03 1.25 1.88 1.63 1.59 9-Nov-03 3 4.13 2.38 3.17 8-Dec-03 8.88 5.63 3.13 5.88 12-Jan-04 8 5.5 4.63 6.04 13-Feb-04 9.25 8.13 5.25 7.54 12-Mar-04 10.25 5.13 4.75 6.71 Mean 6.77 5.07 3.63 Sig. level between harvest time means P< 0.001 Sig. level between site means P< 0.001 Sig. level between harvest time by site means P< 0.001 Least significant difference (lsd) between harvest time means at above sig. level = 2.80 lsd between site means at above sig. level = 1.98 lsd between harvest time by site means at above sig. level = 2.84

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Mean plant heights at FRDS were low compared with those at Kindred and Werrin Farms (Table 4) and site means were inversely correlated with plant age. However, site mean values may have reflected variations in plant populations between the sites, with those at FRDS being the lowest and those at Werrin Farms being the highest populations.

Table 4. Mean individual plant heights (mm) at three sites and six harvest dates of Astragalus in 2003-04

Harvest date FRDS Kindred Werrin Farms Mean 9-Oct-03 79.5 212.8 156.6 149.64 9-Nov-03 148.8 717.3 505.6 457.23 8-Dec-03 484.6 875.6 1167.4 842.53 12-Jan-04 1003.8 1170.8 1759.9 1311.5 13-Feb-04 1271.8 1551 1821.5 1548.1 12-Mar-04 1233 1404 1694.1 1443.7 Mean 703.58 988.58 1184.18 Sig. level between harvest time means P< 0.001 Sig. level between site means P< 0.001 Sig. level between harvest time by site means P< 0.001 lsd between harvest time means at above sig. level = 227.41 lsd between site means at above sig. level = 160.8 lsd between harvest time by site means at above sig. level = 393.89

Plant growth was initially slower at FRDS than at Kindred and Werrin Farms in October and November, 2003 but top-growth of individual plants reached greater values later in the season (Table 5). Thus, top-growth was correlated with stem number rather than with plant height. Table 5. Mean dry weights of top-growth of individual plants (g) at three sites and six harvest dates

of Astragalus in 2003-04.

Harvest date FRDS Kindred Werrin Farms Mean 9-Oct-03 1.44 7.40 4.05 4.30 9-Nov-03 6.69 61.00 25.91 31.20 8-Dec-03 49.85 58.37 82.75 63.66 12-Jan-04 143.09 85.47 98.84 109.13 13-Feb-04 240.82 104.86 109.72 151.80 12-Mar-04 148.25 93.60 68.87 103.57 Mean 98.36 68.45 65.02 Sig. level between harvest time means P< 0.001 Sig. level between site means P< 0.05 Sig. level between harvest time by site means P< 0.001 lsd between harvest time means at above sig. level = 58.56 lsd between site means at above sig. level = 211.03 lsd between harvest time by site means at above sig. level = 101.43

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The mean weight of dried roots taken from individual plants was highest at FRDS and lowest at the Werrin Farms site, again reflecting plant age (Table 6). The values reflected well the number of effective growing seasons elapsed for the plants at each site.

Table 6. Mean dry weights of plant roots (g) at three sites and six harvest dates of Astragalus in 2003-04.

Harvest date FRDS Kindred Werrin Farms Mean 9-Oct-03 28.47 18.43 11.28 19.39 9-Nov-03 41.31 29.11 7.8 26.07 8-Dec-03 31.65 27.29 19.67 26.20 12-Jan-04 61.3 39.18 22.62 41.03 13-Feb-04 117.93 59.95 36.46 71.45 12-Mar-04 131.57 68.81 35.72 78.70 Mean 68.71 40.46 22.26 Sig. level between harvest time means P< 0.001 Sig. level between site means P< 0.001 Sig. level between harvest time by site means P< 0.001 lsd between harvest time means at above sig. level = 26.78 lsd between site means at above sig. level = 18.94 lsd between harvest time by site means at above sig. level = 32.81

The mean length of roots of individual plants (Table 7) reflected the above values for root dry weight.

Table 7. Mean length of roots per plant (mm) at three sites and six harvest dates of Astragalus in 2003-04.

Harvest date FRDS Kindred Werrin Farms Mean 9-Oct-03 460.25 287.25 277 341.50 9-Nov-03 481.25 368.5 284 377.92 8-Dec-03 493.25 376.75 403.38 424.46 12-Jan-04 454.38 324.25 369.88 382.84 13-Feb-04 570.25 524 301.63 465.29 12-Mar-04 600.13 417.63 369.5 462.42 Mean 509.92 383.06 334.23 Sig. level between harvest time means P< 0.01 Sig. level between site means P< 0.001 Sig. level between harvest time by site means – n.s. lsd between harvest time means at above sig. level = 91.10 lsd between site means at above sig. level = 83.03

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The levels of Astragalosides 1, 2 and 4, individually and in total (% dry weight), in roots harvested between October 2003 and February 2004 at the three sites contents are shown in Tables 8, 9, 10 and 11 respectively.

Table 8. Levels of Astragaloside 1 at three sites and six harvest dates in 2003-04.

Harvest date FRDS Kindred Werrin Farms Mean 9-Oct-03 0.236 0.292 0.265 0.264 9-Nov-03 0.272 0.245 0.296 0.271 8-Dec-03 0.298 0.222 0.210 0.243 12-Jan-04 0.324 0.218 0.218 0.253 13-Feb-04 0.253 0.202 0.231 0.229 Mean 0.277 0.236 0.244 0.252 Sig. level between harvest time means - n.s Sig. level between site means P< 0.025 Sig. level between harvest time by site means P< 0.025 lsd between site means at above sig. level = 0.034 lsd between site x harvest time means at above sig. level = 0.077

Table 9. Levels of Astragaloside 2 at three sites and six harvest dates in 2003-04.

Harvest date FRDS Kindred Werrin Farms Mean 9-Oct-03 0.124 0.095 0.074 0.098 9-Nov-03 0.128 0.063 0.078 0.090 8-Dec-03 0.131 0.075 0.057 0.088 12-Jan-04 0.072 0.057 0.050 0.060 13-Feb-04 0.078 0.050 0.051 0.060 Mean 0.107 0.068 0.062 0.079 Sig. level between harvest time means - P<0.001 Sig. level between site means P<0.001 Sig. level between harvest time by site means P< 0.05 lsd between harvest time means at above sig. level = 0.024 lsd between site means at above sig. level = 0.019 lsd between site x harvest time means at above sig. level = 0.025

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Table 10. Levels of Astragaloside 4 at three sites and six harvest dates in 2003-04.

Harvest date FRDS Kindred Werrin Farms Mean 9-Oct-03 0.035 0.003 0.013 0.017 9-Nov-03 0.041 0.021 0.002 0.022 8-Dec-03 0.046 0.013 0.000 0.020 12-Jan-04 0.028 0.017 0.000 0.015 13-Feb-04 0.021 0.010 0.003 0.011 Mean 0.034 0.013 0.003 0.017 Sig. level between harvest time means - n.s Sig. level between site means P<0.001 Sig. level between harvest time by site means P<0.01 lsd between site means at above sig. level = 0.010 lsd between site x harvest time means at above sig. level = 0.170

Table 11. Total levels of Astragalosides 1, 2 and 4 at three sites and six harvest dates in 2003-04.

Harvest date FRDS Kindred Werrin Farms Mean 9-Oct-03 0.395 0.390 0.352 0.379 9-Nov-03 0.441 0.329 0.376 0.382 8-Dec-03 0.476 0.310 0.267 0.351 12-Jan-04 0.424 0.292 0.268 0.328 13-Feb-04 0.352 0.262 0.285 0.300 Mean 0.418 0.317 0.310 0.348 Sig. level between harvest time means - P<0.01 Sig. level between site means P< 0.001 Sig. level between harvest time by site means - n.s lsd between harvest time means at above sig. level = 0.113 lsd between site means at above sig. level = 0.065

Levels of astragalosides in root samples from the three sites were high compared with published data, which result is positive given the standard compounds available for use in these analyses, discussed above. These relatively high levels found are also encouraging from the point of view that relatively young plant material was analysed. The age of plant material is specified only infrequently in published literature but commercial harvests in China may not occur until plants are at least six or seven years old. This deduction is supported by a comparison of the data above across the three sites. Levels of astragalosides from FRDS, where the oldest plants were harvested, were significantly higher than those in plant roots from the other two sites. Additionally, the mean levels of astragalosides were lowest at the site where plants were youngest (Werrin Farms). This trend was present in the data depicting Astragalosides 2, 4 and in the level of total astragalosides.

19

Levels of Astragaloside 1 were highest and those of Astragaloside 4 the lowest at all sites and harvest times, which is consistent with published values. Astragaloside 4 was absent or undetectable in some samples. Levels of Astragaloside 2 varied significantly with harvest time and this was reflected in total astragalosides levels. Levels of Astragalosides 1 and 4 were also lowest in those roots harvested in February. Significant interactions between site and harvest time in levels of astragalosides, reflected a variation on this trend at some sites, where levels first rose and then fell over the five month harvest period, in contrast to other situations where levels declined consistently over this harvest period.

Astragalus

20

5. Bilberry investigations Acquisition of plant material Plant material was requested from several companies and seed banks globally and four lines (from Macedonia, Canada, B&T Seeds in France and Lithuania) were acquired between July 2001 and February 2002. The Macedonian and Canadian materials were received as dried fruits. The extracted seed could not be germinated successfully, notwithstanding the use of a range of conditions and supplements. Given the lengthy delays in, and high costs of, importing vegetative material into Tasmania from overseas (over one year in a quarantine greenhouse and over $1000 per plant for analyses), sourcing of vegetative material was restricted to a search within Tasmania, the results of which were negative. An additional seed-lot was acquired from B&T Seeds in France in 2003 and stratification of this seed was investigated as part of a research student’s project. This showed that low temperature stratification improved germination. However, such germination was still slow, with improvements in percentages still occurring after six months. Early growth Seed was sown on paper and seedlings transferred to pots. Seeds were also sown in small cell trays and rock-wool, in attempts to improve early growth but such growth of germinated seeds under greenhouse conditions was poor and simple tests were carried out in attempts to improve this. Potting media with reduced pH (< 4.5) improved seedling growth but required regular amendment to maintain acidity. Responses to additional nutrients were not found in simple tests, with the exception of one case where improved growth resulted from addition of calcium. Propagation Methods of vegetative propagation were investigated as a response to the above poor seed germination and early growth. Culture of leaf sections, excised buds and stem sections with two nodes was attempted in full strength Murashiga and Skoog media (Murashiga and Skoog, 1962) with 5.5g/L Difco Bacto-agar medium at pH 5.8, after de-contamination with 2 per cent sodium hypochlorite solution for five minutes and 70 per cent ethanol for 10 minutes. While stem sections survived and grew better than leaf sections or excised buds, such growth was again slow and plant mortality was ongoing. As seedling plants increased in size in greenhouse pots and initiated branches from a crown (e.g. 150-200 mm in height after eighteen months), layering of the lowest-borne branches proved to be a simple and effective method of propagation. Stem suckers also appeared on older plants and provided propagules. Field plantings Methods Additional sowings of viable seed in January 2002 allowed larger numbers of seedlings to be produced and over 1000 seedlings, ranging in height from three to six cm, were potted on in November 2002. These were transferred, in the winter of 2003, to two field sites, a nursery paddock at FRDS and a private property near Waratah (lat. 41° 27min S, long. 145° 32min E; elev. 615 m; ann. rainfall 2199 mm), approximately 50 km south of Burnie in Tasmania, where chill factor was likely to be high. Four hundred and ten B&T seedlings and 84 Lithuanian seedlings were transplanted into an area of 150 sq m at FRDS on 29 May 2003, after soil acidification with 15 t/ha of flowers of sulphur and incorporation of 200 kg per ha of single superphosphate. Six hundred and seventy-six B&T and 156 Lithuanian seedlings were transplanted into an area of 250 sq m at Waratah on 10 July 2003, after incorporating 400 kg per ha of

21

3:6:8 mixed fertiliser. This area had recently been cleared of broom, Cytisus scoparius, and the soil’s unmodified pH was 4.4. At each of these sites, eight plants were randomly selected, marked and their growth monitored by monthly measurements of the number and length of all stems. This was done on six occasions between October 2003 and March 2004. Results and discussion The number and mean length of main stems, and the total length of all stems, branches and sub-branches of the eight, randomly-selected plants measured at FRDS during the period October 2003 to March 2004 are shown in Table 12. Table 12. Number and mean length of main stems, and the total length of all stems, branches and

sub-branches of eight bilberry plants at FRDS between October 2003 and March 2004. Date Plant Main

stems

Mean lgth of main stems (mm)

Lgth all stems, branches,sub-branches (mm)

Date Plant Main stems

Mean lgth of main stems (mm)

Lgth all stems, branchessub-branches (mm)

27 Oct 03 1 5 41.6 433 29 Jan 04 1 5 68.4 1192 2 6 41.8 279 2 6 74.3 1020 3 6 49.7 534 3 9 43.1 1423 4 5 63.6 543 4 6 76.5 1568 5 4 30.8 231 5 8 45.5 621 6 4 42.0 334 6 12 56.8 1037 7 5 52.4 267 7 8 63.1 874 8 4 24.3 122 8 0 0 357 27 Nov 03

1 5 44.6 763 27 Feb 04 1 7 81.8 1316

2 6 54.7 797 2 10 58.1 1121 3 7 50.6 1346 3 11 50.5 1747 4 5 81.4 1423 4 8 53.9 1640 5 4 43.8 692 5 7 54.9 1039 6 3 64.0 878 6 8 80.4 1071 7 5 63.6 499 7 8 70.4 918 8 4 32.6 243 8 0 0 414 24 Dec 04 1 5 55.8 1008 31 Mar 04 1 5 92.0 1252 2 6 56.7 1051 2 10 63.2 1251 3 7 58.3 1639 3 11 57.2 1657 4 5 87.8 1550 4 8 69.4 1719 5 4 46.8 760 5 6 72.5 1281 6 3 68.0 949 6 7 69.9 757 7 5 72.6 580 7 8 96.1 1286 8 7 25.1 348

8 3 22.3 467 The number and mean length of main stems, and the total length of all stems, branches and sub-branches of the eight, randomly-selected plants measured at Waratah during the period October 2003 to March 2004 are shown in Table 13.

22

Table 13. Number and mean length of main stems, and the total length of all stems, branches and sub-branches of eight bilberry plants at Waratah between October 2003 and March 2004.

Date Plant Main

stems

Mean lgth of main stems (mm)

Lgth all stems, branches, sub-branches (mm)

Date Plant Main stems

Mean lgth of main stems (mm)

Lgth all stems, branchessub-branches (mm)

22 Oct 03 1 10 39.2 562 29 Jan 04 1 10 50.4 1116 2 7 52.9 540 2 12 73.5 2425 3 5 56.0 395 3 5 50.2 377 4 11 60.8 679 4 12 86.2 2298 5 8 71.9 575 5 12 76.9 2147 6 7 36.6 366 6 10 59.1 1636 7 4 57.8 231 7 6 80.3 1139 8 3 77.3 372 8 9 81.6 1110 26 Nov 03

1 10 67.8 1834 24 Feb 04 1 14 68.8 2390

2 7 87.3 1656 2 13 68.8 1924 3 5 63.4 728 3 2 80.0 216 4 11 78.5 2162 4 13 86.7 2217 5 8 90.0 1612 5 12 86.4 2410 6 7 57.9 904 6 10 73.2 1934 7 4 98.5 743 7 8 76.9 1294 8 3 100.0 540 8 9 91.9 1244 19 Dec 04 1 10 67.8 1809 31 Mar 04 1 14 77.4 2664 2 9 80.3 2065 2 13 69.6 2138 3 5 63.6 526 3 2 81.5 3232 4 11 80.3 2219 4 13 90.0 2372 5 11 73.6 1792 5 12 91.0 2711 6 9 52.2 1167 6 11 77.5 2367 7 5 84.0 870 7 11 73.5 1530 8 6 63.7 679

8 9 76.8 1325 A factorial analysis of variance applied to the above site by time data (Table 14) showed that growth rate of bilberry at Waratah was significantly (P< 0.001) greater than that at FRDS but that the interaction between site and time of record was not significant. Flowering commenced on some plants in September 2004, when plants were 32 months old. Reflecting the better growth at Waratah compared with FRDS, flowers were more frequent on plants at Waratah (Table 15). A small number of fruit was set at Waratah but no fruit set occurred at Forthside.

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Table 14: Mean values (per plant, mm) of the total length of all stems, branches, sub-branches on eight bilberry plants at FRDS and Waratah over the period October 2003 to March 2004.

Date Mean total length of all

stems, branches and sub-branches (mm)

Site Mean total length of all stems, branches, sub-

branches (mm) 22/27 Oct 2003 403.9 26/27 Nov 2003 1051.3 FRDS 922.9 19/24 Dec 2003 1188.2 Waratah 1442.5 29 Jan 2004 1271.3 24/27 Feb 2004 1430.9 31 Mar 2004 1750.6 P level <0.01 P level <0.001 Mean 1182.7 Mean 1182.7 lsd at above P level 272.5

lsd 359.8

Table 15. Percentages of flowers present on plants at Waratah and Forthside in season 2004-05 (the first flowering season).

Date Location 2 October 2004 29 September 2004 26 November 2004 5 February 2005 Waratah 4.6 37.4 3.4 8.8 FRDS 8.2 14.2 0 2.6 Temperature and shading investigations As part of a student project, carried out in conjunction with this work, eight, potted bilberry plants were placed in each of three cool rooms set at 4ºC, 8ºC and 12ºC respectively. The plants were placed within light-boxes each having a 16 hr light/8hr dark, diurnal regime. In the 8ºC room, an additional box with an 8 hr light/16hr dark regime was added. Plant growth was monitored. While regime conditions were not maintained after 2 months, measurements indicated that growth rate was positively correlated with temperature and some growth continued at 4ºC. However, leaves of plants in 4ºC turned red/brown. No significant differences in growth were found between the two daylength regimes at 8 ºC. Information gained from overseas study (see below), suggested that the growth effects of shading should be investigated. Methods Ten plants growing at the Waratah site, therefore, were randomly selected, tagged and shaded by surrounding with a shadecloth, which halved transmitted light. A further ten, randomly-selected plants were tagged but not shaded. Between September 2004 and February 2005, the growth of these plants was individually scored as previously described (total length of all stems, branches, sub-branches). Results and discussion The mean values for the ten shaded and un-shaded treatment plants for the six scoring dates are shown in Table 16.

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Table 16. Mean values (per plant, mm) of the total length of all stems, branches, sub-branches on ten shaded and ten un-shaded bilberry plants at Waratah over the period September 2004 to

February 2005.

Date Mean total length of all stems, branches, sub-branches (mm) Shaded Un-shaded Mean 2 September 2004 160.5 242.0 201.3 4 October 2004 171.5 261.5 216.5 29 October 2004 200.5 272.5 236.3 22 November 2004 256.0 278.0 267.0 2 February 2005 433.0 434.0 433.5 Mean 244.3 297.6 271.0 lsd

(P=0.05) 40.8 (P=0.001)

110.5 These data show that, while those plants, which were randomly selected for shading were significantly smaller (P=0.05) on average, than those selected for un-shaded comparison, these plants increased in size by 170 per cent during the period, compared with the increase in size of 79 per cent attributed to un-shaded plants. Overseas study The author travelled to Poland between 9-13 July 2003, during alternative studies, to learn more of the growth and wild harvesting of bilberry. The visit was arranged through a previous acquaintance, Professor Zenon Weglarz, who is Head of the Department of Vegetable and Medicinal Plants, in the Faculty of Horticulture and Landscape Architecture at Warsaw Agricultural University. The Department had twelve academic staff and a similar number of research higher degree students (RHDs) and its main research areas were in medicinal plant development, improvement of essential oil crops, vegetable physiology and agronomy and new cultivar evaluation. Teaching and laboratories were centred at Ursynow, in southern Warsaw, where much new construction was underway. There were extensive field experimentation facilities at several centres including a large collection of medicinal plants. While this Department itself was not large, the Faculty also comprised Departments of Environmental Protection, Landscape Architecture, Ornamental Plants, Plant Genetics and Biotechnology, Plant Pathology, Pomology, Applied Entomology and a Horticultural Economics Section. It had 260 academic staff and research students. However, only one postgraduate was from overseas and there were strong drives to improve international linkages, fuelled particularly by the prospect then of Poland’s entry into the European Union. One day was spent examining stands of bilberry in the forest understorey in the Podlaski region of eastern Poland, some 200 km from Warsaw and about 30 km from the Russian border. Professor Weglarz and the author were accompanied by Miroslaw Angielczyk, who operated a business buying, processing and re-selling a range of medicinal herbal products. Mr Angielczyk had also recently completed an MSc in the above Department and started a PhD on Hierochloe spp as a source of coumarin. We examined bilberry stands at several sites, where the species formed a carpet-like understorey of up to 40 cm in height, beneath both deciduous (Betula, Quercus, Fagus spp.) and coniferous trees (Pinus, Picea spp), together with alder and juniper. The best stands of bilberry were in about fifty per cent shade. Bilberry was sparse or absent where deciduous species dominated and produced higher levels of shading. It was also sparse or absent in un-shaded areas. Soils were acid sands over clay and bilberry top-growth was produced from long, prostrate adventitious roots, which were near the soil surface but beneath a deep layer of leaf litter and humus. While we met a few people still gathering the remaining fruit, the majority of the wild crop had been collected earlier as a result of the unusually dry seasonal conditions. The season’s crop was considered to be light, the fruit small and prices relatively high. However, fruit was still being sold on the

25

roadside for 6.50 Zlotych (then A$ 2.75) per kg. Picking the fruit, which was about 8 mm in diameter and dispersed on the bush, by hand appeared to be difficult and slow, and small, hand-held trays with comb-fronts were sometimes used. While these were said to be illegal in some areas due to their destructive action on the plant, there is a small trade in the leaf itself (in the order of a few tonnes) as an anti-diabetic remedy and the author is now aware of one company in Holland investigating the activity of compounds found in leaf extract (arbutin and other phenolics). Other plant materials from the forest and surrounding areas, traded by Mr Angielczyk included alder (a purgative), Epilobium (a remedy for prostate enlargement?), Achillea, Hypericum, Rubus, Heracleum and Crateagus and Sambucus. On the following day the author was introduced to Dr Stanislaw Glowacki, a recently retired, honorary researcher, who had investigated bilberry for several years, albeit from the forest ecology viewpoint. While the discussions were conducted via Prof. Weglarz as interpreter, some important points emerged regarding the wild crop in Poland and supporting literature was obtained. Poland has approximately 28 per cent forest cover and about 11 per cent of this contains bilberry. While collection and export of the fruit was a major industry a generation ago (26,000 tonnes were exported in 1966), collection had dropped to around 10,000 tonnes and export (mostly of fresh fruit) was then down to around 2,000 tonnes due to declining yield and poor organisation of companies. Most export was to Germany. In addition to fresh sales and medicinal preparations, fruit extracts were used to colour white wine for sale as red wine, as the components are comparable. Forest populations of bilberry increase slowly with around 14 per cent cover gained after fifty years. In lowland areas, propagation and fruiting only progressed well when shade reached 40-50 per cent but at higher altitudes, plants will grow and fruit in full sun. Above 500 metres altitude in Poland the species does not fruit in shade and fruit size is improved (marginally) at higher altitudes (500-1000m). It is very tolerant of windy conditions. While there was considered to be variation among genotypes with regard to actives and genetic variation in Vitamin C had been shown, contents of phenolics had not yet been the subject of much investigation. This brief study was valuable in developing the work on bilberry reported here.

Bilberry

26

6. Feverfew investigations A line of feverfew seed was acquired by BRA and sown in a field planting on the company’s property in North-west Tasmania in November 2001. This sowing resulted in a low germination but established plants grew well under regular irrigation. Samples of plant tops (20cm of stem when first flowers opened) were taken for analysis in January 2002 (late flowering due to late sowing) and were sent to a contact company (Ivax Pharmaceuticals S.R.O., Czech Republic), which had previously shown interest. The company returned analyses of 4.78 mg/g parthenolide in a sample with 25 per cent of flowers open and of 5.47 mg/g in a sample with 75 per cent of flowers open). These values were said to compare favourably with those from material generally handled by this company (range of 1.2 – 4.4 mg/g parthenolide). While the company was pleased with the high quality of the samples, it indicated that trading in feverfew at that time had stopped due to oversupply. Variety comparison Methods The project sought seed lines of feverfew from Australian and overseas suppliers in October and November 2001. Four lines were received prior to May 2002 (Austral, B&T World Seeds, B&T World Seeds organic and Richters) and a fifth line, “Golden”, was received in December 2002. Seed of these lines was germinated in greenhouse trays, re-potted and transplanted into a replicated field experiment from October 2002, with lines received later being accommodated in the field layout. The experiment was carried out at FRDS. Five replicates of the five lines (treatments) were laid out in randomized blocks and each plot (12 metres long by 1.6 metres wide) consisted of three rows each containing 24 plants. A mixed fertilizer was applied before transplanting and plots were irrigated as required. The trial was trimmed mechanically after flowering in February 2003, weeded as required and maintained through winter 2003. The plants proved hardy to winter conditions, although some leaf damage occurred in June, July and August 2003, particularly in the “Golden” line. This damage was diagnosed as being caused by Septoria sp. and responded to applications of Kocide DF ® fungicide. The trial was maintained in a similar manner through 2003, with the additional application of Fusilade ® herbicide to control grass weeds. Flowering plants were again trimmed mechanically on 13 January 2004 and, on 7 April 2004, plant re-growth was harvested from each plot, with young shoot growth (approx. top 200mm) and the remainder of the above-ground plant parts separated and weighed. Sample material of the top growth fraction only was oven-dried at 40°C, ground in a laboratory hammer mill and provided to BRA laboratories for analysis of parthenolide. This was carried out according to the method developed at these laboratories, based on Anon., (2000). Yields of plant material and parthenolide from top-growth were calculated for each plot and analyses of variance carried out. Result s and discussion Treatment yields of total (top-growth plus balance of above-ground parts) dry matter and dry top-growth (approx. 200mm) per hectare of the five varieties are shown in Table 17. Parthenolide contents in top-growth and the calculated yields of parthenolide in top-growth per hectare of the five varieties are shown in Table 18.

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Table 17. Total dry matter yield (kg/ha) and top growth (approx. 200mm) dry matter yield (kg/ha) of five feverfew varieties, April 2004.

Variety Total dry matter yield

(kg/ha) Top growth (approx. 200mm)

dry matter yield (kg/ha) Austral 6352 1333 B&T (not organic) 7633 1512 B&T (organic) 7276 1349 Golden 1661 838 Richters 6708 1350 Mean 5926.0 1276.7 lsd P=0.05 304.2 306.5 lsd P=0.01 422.3 lsd P=0.001 576.2

Table 18. Parthenolide contents (mg/g dry matter) in top growth (approx. 200mm) and extrapolated yields of parthenolide in top growth (kg/ha) of five feverfew varieties, April 2004.

Variety Parthenolide in top growth (mg/g dry matter)

Calculated parthenolide yields from top growth (kg/ha)

Austral 18.17 24.30 B&T (not organic) 21.33 32.09 B&T (organic) 3.61 4.93 Golden 25.45 21.08 Richters 17.17 23.25 Mean 17.15 21.13 lsd P=0.05 2.69 6.28 lsd P=0.01 3.72 8.66 lsd P=0.001 5.11 11.90

Seasonal growth analysis Methods At the time of establishment of the variety trial described above, an adjacent area was planted in a similar manner with seedlings of the variety Austral. These were maintained with identical and similarly-timed field operations to those used on the variety trial, with the exception that flowering plants were not trimmed mechanically in January 2004. In order to gain information on the possible fluctuation of parthenolide content in top-growth over the 2003-04 spring/summer growth period, eight individual plants were selected randomly from this Austral planting on each of five occasions (14 October 2003, 14 November 2003, 10 December 2003, 21 January 2004 and 16 February 2004) and destructively harvested. At each harvest, the height of each plant was recorded, as were the number of flower buds, open flowers and spent flowers present. The individual plants again were divided into top-growth (approx top 200mm) and the balance of above ground parts. Plant material was dried, weighed and ground as described

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previously and both samples of top-growth and the balance of above-ground parts analysed for parhenolide content. Results and discussion Table 19 shows the mean values of plant height, flower buds, open flowers and spent flowers from the eight plants destructively harvested on each of the harvest occasions. Table 20 shows the mean dry weight of top growth (approx. 200mm) and of the remainder of the above-ground parts for the eight plants destructively harvested on each of the harvest occasions, together with the mean parthenolide concentrations in these fractions of dry plant dry matter.

Table 19. Mean plant height, numbers of flower buds, open flowers and spent flowers from eight plants destructively harvested on each of the harvest occasions.

Harvest date Mean plant

height (mm) Mean no. flower

buds Mean no. open

flowers Mean no. spent

flowers 14 October 2003 280.0 0.6 2.5 0.0 14 November 2003 525.5 6.3 9.4 0.0 10 December 2003 735.6 535.9 294.1 34.4 21 January 2004 706.6 0.0 18.9 1760.6 16 February 2004 769.1 9.1 13.1 1141.5 Mean 603.4 110.4 67.6 587.3 P < 0.001 < 0.001 < 0.001 < 0.001 lsd at sig.level shown 95.64 154.10 125.67 657.01

Table 20. Mean dry weight of top growth and of the remainder of the above ground parts for

the eight plants destructively harvested on each of five dates, together with the mean parthenolide concentrations in these fractions.

Harvest date Mean dry wt of

top growth (g) Mean

parthenolide conc. in top

growth (mg/g)

Mean dry wt of remainder of above ground plant parts(g)

Mean parthenolide

conc. in remainder (mg/g)

14 October 2003 29.4 7.06 106.3 5.16 14 November 2003 31.5 5.01 168.0 3.71 10 December 2003 63.5 4.44 195.1 3.42 21 January 2004 101.0 4.54 150.5 3.16 16 February 2004 71.6 2.90 143.7 2.94 Mean 59.4 4.79 152.7 3.68 P < 0.001 < 0.001 < 0.01 < 0.001 lsd at sig.level shown 45.10 0.947 61.10 1.183

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The five lines compared in the variety trial returned total dry matter yields which were significantly different (P<0.001, Table 17), the largest differences being due to the dwarf habit of the cultivar “Golden”. However, B&T lines were also significantly greater (P<0.05) than other cultivars with respect to dry matter yield. The dwarf habit of the cultivar “Golden” was also reflected in the low yield of top growth dry matter. Concentrations of parthenolide found in the top growth of four of the five varieties were very high (Table 18) in comparison to those found in the harvest samples of variety Austral carried out in sequential growth analysis (Table 20) and also in comparison to those values quoted by the company contacted earlier in the project. This suggested that the flush of new growth sampled after the plants had been trimmed mechanically on 13 January 2004 was particularly rich in parthenolides. These levels led to high values for calculated parthenolide yields per hectare. The concentration of parthenolide in the variety “Golden” was significantly higher (P<0.01, Table 18) than that in all other varieties, although insufficiently so to compensate for the variety’s low dry matter yield. However, the fifth variety tested, B&T organic, while returning high yields of dry matter, contained low levels of parthenolide in both top growth and in all above ground plant parts. Sequential sampling of the variety Austral has shown that concentrations of parthenolide in both top growth and the balance of above ground plant material were high at the beginning of seasonal growth in October but fell as plant growth accelerated during the spring and summer (Table 20). Concentrations in top growth were consistently higher than in the balance of above ground material.

Feverfew

30

7. Stevia investigations The work undertaken on stevia in this project was coordinated with that carried out by Professor David Midmore and Andrew Rank at Central Queensland University (CQU). A meeting was held at CQU in December 2001, at which Andrew Rank reported on a study tour to investigate stevia production and interest in a proposal to RIRDC to fund work on stevia was confirmed. The subsequent proposal by Professor Midmore to RIRDC was approved in March 2002 and a site in North-west Tasmania was included in several selected to investigate the potential of stevia in different latitudes in eastern Australia. A progress meeting was held in northern New South Wales in November 2002. Further Stevia rebaudiana genotypes were received by the Tasmanian project from Andrew Rank and samples returned for analysis. Acquisition of lines Five lines of stevia seed were acquired at the start of the project in November 2001 (from Brazil, Richters in Canada, B&T Seeds in France, and from New Gippsland Seeds and the CQU project (cv Zhang Jingong) in Australia). These lines germinated poorly. In March 2002, 120 potted stevia plants were located in a wholesale nursery near Hobart, where they were in poor demand as ornamental plants (Herb Herbert herbs) and in May 2002 some additional lines were supplied by the CQU project. These were Sahin, T2, T4, 94/8 and 83/27. Seed of the lines Sahin, T2 and T4 germinated more readily than did 94/8 and 83/27. Glasshouse propagation Methods All seedlings were grown under ambient, and then supplementary, winter-heated, glasshouse conditions and a range of techniques was used in attempts to propagate them. Tissue culture using both solid and liquid media was attempted. Propagation using stem cuttings from both ‘soft-stem’ and ‘hard-stem’ tissue, usually consisting of two nodes, was attempted. Leaves were removed from the base of the cuttings and two to four leaves were retained at the apex, as fewer apical leaves were found to be inadequate for growth. Sub-division of mature plant crowns was also carried out. Results and discussion Tissue culture using solid media provided more consistent results than did liquid media. Lines differed in their readiness to strike roots from such cuttings with 94/8, Brazil and Richters being the easiest. However, even with these lines, roots were not apparent for three to four weeks after preparation of the cuttings. The practice generally proved unsuccessful in winter months, with October to February being optimal. Sub-division of mature plant crowns proved to be the simplest and most efficient way of propagating the material, with at least ten splits usually obtained from one mature crown and new growth evident after one to two weeks.

31

Seed collection and germination In April and May 2003, plant stems of all lines were individually bagged before flower buds opened and seed collected after shedding. Seed was sown in trays containing seed-raising mixture and placed on a hot bed within a glasshouse with supplementary overnight heating. Seed of the lines Sahin, T4 and Richters germinated more readily than seed of other lines, although it was necessary to select well-formed seed with a dark seed coat to maximize germination. Seeds with a light-coloured coat germinated poorly. Initial field planting, March 2002 Methods The potted stevia plants located in a wholesale nursery near Hobart were planted into the field in March 2002, notwithstanding the advanced season, in order to expedite investigations with the species and, at least, to test the plants’ ability to survive local winter conditions. Plants from this source were also maintained in the glasshouse to provide propagation material. Results and discussion These plants grew well until mid-April, when top-growth rapidly senesced. In the following spring (October 2002), about 40 per cent of these plants re-grew from the crown. The presence of sclerotinia and other diseases during the intervening winter may have contributed to this low level of regeneration. Field trial planting, November 2002 Methods In November 2002, glasshouse-propagated stem cuttings of the above five stevia lines (few plants were available of the B&T line) were planted out at FRDS, near Devonport in Tasmania, into a krasnozem soil (pH 6.1). Each plot was 12 m long by 1.6 m wide and consisted of two rows of plants at a density of ten per sq m. Treatment lines were replicated where sufficient planting material was available but this was not possible with the Richters, New Gippsland Seeds and Zhang Jingong lines. A mixed fertilizer was applied before transplanting. Woven, plastic weed matting was placed to assist weed control and plots were irrigated as to a 35 mm deficit as required. The weed matting contributed to poor irrigation of the plots initially and was replaced by pea straw mulch in February 2003. In March 2003, sclerotinia was observed in the plots and some plants succumbed to the disease, notwithstanding application of Sumisclex 500® fungicide, and the pea straw mulch was removed. One whole plant from each plot was harvested (to 10cm above ground level) on 29 January, 21 February and 7 April 2003 and (at the first harvest) hung in an outdoor, sheltered area to air-dry. This process worked poorly in overcast conditions and later harvests were oven-dried at 40°C. Dried leaf and stem were segregated and weighed and leaf samples provided to BRA and the CQU project for analysis. Flowering heads of individual plants in the trial were bagged for seed collection when flowering occurred between mid January and late March 2003, with the exception of the New Gippsland Seeds line which

32

produced few, very late flowers. Plots were maintained through winter 2003 but few plants of any of the six treatment lines survived into Spring 2003, with plants remaining in four plots only. This new season’s growth on these plants first appeared above the ground in late October 2003. These plants were dug from the field plots in January 2004, returned to the glasshouse and used for further propagation. Results and discussion Field trial planting, November 2002 Table 21 shows the dry weight of single plants of the five lines collected for leaf sample analysis on three occasions. Table 21. Mean dry weight of single plants of five stevia lines collected for leaf sample analysis on

three occasions in 2003. Harvest date Line Herbert herbs Zhang Jingong Brazil New Gippsland Richters Plant dry wt (g) Plant dry wt (g) Plant dry wt (g) Plant dry wt (g) Plant dry wt (g) 29 Jan 03 8 6 6 3 4 21 Feb 03 46 82 107 47 62 7 April 03 111 86 290 86 206 Leaf material was consistently close to 50 per cent of total plant weight. A verbal report from the CQU project indicated that the New Gippsland line was particularly rich in stevioside. Few plants of any of the six lines survived the winter in 2003, with the four remaining plots each containing less that ten plants each. The new season’s growth on these plants appeared above the ground in late October 2003. Expanded field plantings, 2003-04 Methods In October and November 2003, new plantings of propagated material were made at two sites, the first at FRDS, near Devonport, where an alternative paddock with less exposure to wind was selected. A second site was established on a commercial property at Merseylea, inland from Devonport, where summer temperatures usually exceed those of the coastal area. At each of these sites, ten lines (Brazil, Richters, New Gippsland Seeds, Zhang Jingong, Herb Herbert, Sahin, T2, T4, 94/8 and 83/27) were planted, at a density of ten plants per sq m, in single plots, each ten sq m in area. In addition, one further genotype (HER, transplants supplied by the CQU project as part of its investigation over several sites and latitudes in eastern Australia) was planted in a single plot of 100 sq m at each of these sites (at FRDS on 15 October 2003 and at Merseylea on 8 October 2003). Again, a density of ten plants per sq m was used. Plots were fertilized and irrigated as before and weeded by hand. In the case of the line HER, four plants at FRDS and eight plants at Merseylea were marked and their height noted monthly. Flowering dates were also recorded. At each of the two sites, an additional four plants, comprising two lots of two plants each, were destructively harvested each month.

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The fresh and oven dry (60°C) weights of each of the two plants in the first lot were noted, the stem and leaf material segregated, and a sample of leaf ground in a laboratory hammer mill and stored (-15°C) for analysis of stevioside, rebaudioside A and iso-steviol levels in extracts by the BRA laboratory, using high performance liquid chromatography with evaporative light scattering detection, after the method of Kolb et al (2001). The second lot of two plants was dried similarly, ground collectively and a representative sample sent to the CQU project investigators for analysis. In addition, all remaining plants in the plot were harvested to 10 cm above ground level on a date shortly before flowering, the total fresh weight recorded and the dry weight of a twenty-plant sample obtained. The production of dry matter per ha was calculated from these data. Four plants of each of the remaining ten lines were destructively harvested on two occasions and their total fresh and dry weights obtained. A representative sample of leaf was ground for analysis of stevioside, rebaudioside A and iso-steviol levels in extracts, as noted above. Results and discussion Line HER at FRDS Some transplants started to flower on 9 December 2003. Table 22 shows the individual heights of four plants noted on seven occasions during the season.

Table 22. Growth of four HER stevia transplants at FRDS as reflected in individual plant height (cm).

Individual plant height (cm)

Date Plant 1 Plant 2 Plant 3 Plant 4 Mean 16 October 03 20 12 18 14 16.0 17 November 03 21 14 20 18 18.3 16 December 03 24 18 22 26 22.5 16 January 04 40 25 32 38 33.8 16 February 04 65 29 61 57 53.0 16 March 04 82 30 64 62 59.5 13 April 04 110 30 65 83 72.0

The initially single-stemmed transplants began to branch in January and Plants 1-4 had 10,18, 10, 10 main branches respectively on the final date. Sequential destructive harvests. Table 23 shows the height, total fresh weight and dry weight of stem and leaf of four individual plants harvested on six occasions for analyses of extract.

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Table 23. Height, total fresh weight, stem dry weight and leaf dry weight of four HER stevia transplants plants harvested on six occasions at FRDS for analyses of extract.

Individual plant height (cm) Date

Plant 1 Plant 2 Plant 3 Plant 4 Mean 4 December 03 20 22 13 18 18.3 5 January 04 33 27 26 24 27.5 4 February 04 45 34 38 32 37.3 4 March 04 51 40 52 66 52.3 31 March 04 77 69 59 89 73.5 13 April 04 100 52 72 54 69.5

Total fresh weight (g) Date

Plant 1 Plant 2 Plant 3 Plant 4 Mean 4 December 03 8.1 11.3 15.6 14.4 12.4 5 January 04 68.7 120.1 22.2 103.2 78.6 4 February 04 212.6 129.0 137.8 116.4 149.0 4 March 04 337.1 222.2 283.2 234.0 269.1 31 March 04 649.0 531.6 261.2 325.8 441.9 13 April 04 243.4 197.6 175.0 195.4 202.9

Stem dry weight (g) Date Plant 1 Plant 2 Plant 3 Plant 4 Mean

4 December 03 0.5 0.8 0.6 0.8 0.69 5 January 04 4.2 8.4 2.1 7.1 5.45 4 February 04 22.0 13.6 14.0 9.0 14.65 4 March 04 36.1 22.6 31.6 23.6 28.48 31 March 04 96.6 70.2 37.5 39.8 61.03 13 April 04 37.1 40.2 25.9 15.7 29.73

Leaf dry weight (g) Plant 1 Plant 2 Plant 3 Plant 4 Mean

4 December 03 1.3 1.6 2.4 2.0 1.83 5 January 04 11.7 17.2 3.4 14.2 11.63 4 February 04 28.7 14.4 19.4 14.3 19.20 4 March 04 60.3 34.1 41.8 36.6 43.20 31 March 04 90.5 78.3 38.6 46.9 63.58 13 April 04 33.7 35.8 22.9 29.6 30.50

Leaf material from two plants per harvest date was combined for extract analysis in BRA’s laboratories and that from the remaining two plants sent to the CQU project for analysis. Table 24 shows the levels of stevioside, rebaudioside A and iso-steviol found through the BRA laboratories’ analyses.

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Table 24. Stevioside, rebaudioside A and iso-steviol contents of leaf material obtained from sequential harvests of line HER at FRDS between December 2003 and April 2004.

Harvest date Stevioside (%) Rebaudioside A (%) Iso-steviol (%) 4 December 03 2.75 0.83 0.00 5 January 04 4.93 1.71 0.00 4 February 04 7.63 2.25 0.00 4 March 04 6.70 2.42 0.00 31 March 04 4.62 3.44 0.00 13 April 04 4.80 1.99 0.00

The remaining 877 HER plants in this plot (total area = 97.2 sq m) were harvested on 4 March 2004. Their total fresh weight was 217.6 kg. The fresh weight of a 20-plant sub-sample was 6118 g and its corresponding oven dry weight (60°C, 24h) was 1321 g. The production of dry matter per ha, without adjustment for the 24 plants used in sequential, destructive samples, was calculated from these data to be 4834 kg per ha. Line HER at Merseylea Transplants were planted into the field of 10 October 2003. At this time several plants showed symptoms of Botrytis disease and were not transplanted. Flowering commenced on 18 December 2003 but was very variable between plants. Plants grew well but in mid–February dark lesions, due to Botrytis were noted on the older leaves of plants, which symptoms increased over time. Table 25 shows the individual heights of four plants noted on seven occasions during the season.

Table 25. Growth of eight HER stevia transplants at Merseylea as reflected in individual plant height.

Individual plant height (cm) Date

Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Plant 6 Plant 7 Plant 8 Mean 13 Oct 03 21 11 16 24 20 14 14 20 17.5 13 Nov 03 23 14 18 26 20 20 16 28 20.6 12 Dec 03 30 26 23 27 24 21 23 35 26.1 13 Jan 04 45 53 40 35 40 47 48 57 45.6 13 Feb 04 55 66 60 50 63 75 68 75 64.0 12 Mar 04 87 80 73 76 75 88 74 80 79.1 13 Apr 04 103 100 82 110 97 117 90 89 98.5 The initially single-stemmed transplants began to branch in December. Sequential destructive harvests. Table 26 shows the height, total fresh weight and dry weight of stem and leaf of four individual plants harvested from Merseylea on six occasions for analyses of extract.

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Table 26. Height, total fresh weight, stem dry weight and leaf dry weight of four HER stevia transplants plants harvested on six occasions at Merseylea for analyses of extract.

Individual plant height (cm) Date

Plant 1 Plant 2 Plant 3 Plant 4 Mean 18 December 03 35 24 25 28 28.0 19 January 04 40 40 37 39 39.0 19 February 04 64 86 49 59 64.5 19 March 04 107 97 87 85 94.0 31 March 04 98 95 91 120 101.0 13 April 04 94 107 82 109 98.0

Total fresh weight (g) Date Plant 1 Plant 2 Plant 3 Plant 4 Mean

18 December 03 104.8 48.2 34.9 86.4 68.58 19 January 04 113.4 172.5 76.4 48.4 102.68 19 February 04 288.1 195.4 107.8 267.2 214.63 19 March 04 283.1 244.3 312.2 365.1 301.18 31 March 04 215.4 513.2 320.1 412.2 365.23 13 April 04 412.3 456.4 198.8 389.1 364.15

Stem dry weight (g) Date Plant 1 Plant 2 Plant 3 Plant 4 Mean

18 December 03 7.5 3.0 1.8 4.2 4.13 19 January 04 13.3 17.2 5.7 6.3 10.63 19 February 04 29.3 30.2 9.7 31.0 25.05 19 March 04 59.0 41.1 47.1 57.7 51.23 31 March 04 29.4 86.2 47.0 64.4 56.75 13 April 04 61.8 60.9 32.4 63.7 54.70

Leaf dry weight (g) Plant 1 Plant 2 Plant 3 Plant 4 Mean

18 December 03 8.4 4.7 3.7 9.3 6.53 19 January 04 16.4 26.2 11.0 6.6 15.05 19 February 04 37.2 30.8 14.1 43.5 31.40 19 March 04 42.0 43.8 42.0 54.7 45.63 31 March 04 31.3 63.6 51.0 62.8 52.18 13 April 04 60.1 75.9 28.8 55.5 55.08

Leaf material from two plants per harvest date was combined for extract analysis in BRA’s laboratories and that from the remaining two plants sent to the CQU project for analysis. Table 27 shows the levels of stevioside, rebaudioside A and iso-steviol found through the BRA’s analyses.

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Table 27. Stevioside, rebaudioside A and iso-steviol contents of leaf material obtained from sequential harvests of line HER at Merseylea.

Harvest date Stevioside (%) Rebaudioside A (%) Iso-steviol (%) 18 December 03 1.61 0.81 0.00 19 January 04 5.57 1.66 0.00 19 February 04 7.90 2.92 0.00 19 March 04 7.15 3.70 0.00 31 March 04 8.41 4.10 0.00 13 April 04 6.00 2.94 0.00

The remaining 843 HER plants in this plot (total area = 93.6 sq m) were harvested on 11 March 2004. Their total fresh weight was 227.8 kg. The fresh weight of a 20 plant, sub-sample was 6550 g and its corresponding oven dry weight (60°C, 24h) was 1700 g. The production of dry matter per ha, without adjustment for the 24 plants used in sequential, destructive samples, was calculated from these data to be 6317 kg per ha. Other lines at FRDS The ten remaining lines (Brazil, Richters, New Gippsland Seeds, Zhang Jingong, Herb Herbert, Sahin, T2, T4, 94/8 and 83/27) were variable in flowering time, with the majority of plants within these lines commencing flowering between late March and late April 2004. There was little difference between the lines with respect to flowering date, given this within-line variation. Tables 28 and 29 compare the fresh weight of plants, the dry weight of stem and leaf and the levels of stevioside, rebaudioside A and iso-steviol in extracts of samples of these ten lines harvested on the 4 February and 23 February 2004 respectively.

Table 28. Fresh weight, stem dry weight, leaf dry weight and levels of stevioside, rebaudioside A and iso-steviol in extracts of ten stevia lines harvested on the 4 February 2004.

Line Plant fresh

weight (g) Stem dry wt per plant (g)

Leaf dry wt per

plant (g)

Stevioside (%)

Rebaudioside A (%)

Iso-steviol (%)

Brazil 257 34.5 26.2 5.45 5.47 0.00 Herb Herbert 303 36.0 42.0 6.53 3.18 0.00 Richters 272 26.8 34.3 6.05 4.59 0.00 New Gippsland 162 15.5 23.8 10.20 1.42 0.00 Zhang Jingong 219 17.5 33.0 3.06 10.64 0.00 Sahin 221 18.8 21.8 5.29 1.32 0.00 T4 282 25.3 32.0 3.91 2.91 0.00 T2 186 16.3 21.5 5.10 2.49 0.00 94/8 209 19.8 24.0 7.04 0.56 0.00 83/27 116 9.5 15.0 5.80 2.77 0.00

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Table 29. Fresh weight, stem dry weight, leaf dry weight and levels of stevioside, rebaudioside A and iso-steviol in extracts of ten stevia lines harvested on the 23 February 2004.

Line Plant fresh

weight (g) Stem dry wt per plant (g)

Leaf dry wt per

plant (g)

Stevioside (%)

Rebaudioside A (%)

Iso-steviol (%)

Brazil 166.3 23.8 25.8 4.59 3.98 0.00 Herb Herbert 112.5 6.8 16.0 5.44 2.30 0.00 Richters 185.8 10.8 24.3 4.76 2.07 0.00 New Gippsland 189.8 13.8 26.5 9.23 0.59 0.00 Zhang Jingong 158.3 17.8 35.8 5.11 4.92 0.00 Sahin 239.3 11.0 25.5 7.16 1.97 0.00 T4 158.3 14.8 25.5 5.23 2.33 0.00 T2 184.5 11.0 29.3 4.50 3.45 0.00 94/8 181.3 10.3 24.5 6.75 1.39 0.00 83/27 139.8 6.3 21.3 5.75 2.21 0.00 While mean plant fresh weights and mean dry weights of stem and leaf were variable between harvests, largely due to some loss of leaf and stems breaking away from the crown before the second harvest (perhaps in part due to the Botrytis present), significant differences between lines were found in the stevioside and rebaudioside A contents of leaf samples (Table 30).

Table 30. Mean stevioside and rebaudioside A contents of leaf samples of lines, over two harvest times.

Line Stevioside (%) Rebaudioside A

(%) Brazil 5.02 4.73 Herb Herbert 5.99 2.74 Richters 5.41 3.33 New Gippsland 9.72 1.01 Zhang Jingong 4.09 7.78 Sahin 6.23 1.65 T4 4.57 2.62 T2 4.8 2.97 94/8 6.90 0.98 83/27 5.78 2.49 Mean 5.848 3.028 lsd 4.279 (P=0.001) 4.542 (P=0.01)

Other lines at Merseylea

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The ten lines (Brazil, Richters, New Gippsland Seeds, Zhang Jingong, Herb Herbert, Sahin, T2, T4, 94/8 and 83/27) were again variable in flowering at the Merseylea site, with most flowering in late March through early April 2003. Tables 31 and 32 compare the fresh weight of plants, the dry weight of stem and leaf and the levels of stevioside, rebaudioside A and iso-steviol in extracts of samples of these ten lines harvested on 11 February and 24 February 2004 respectively.

Table 31. Fresh weight, stem dry weight, leaf dry weight and levels of stevioside, rebaudioside A and iso-steviol in extracts of ten stevia lines harvested on the 11 February 2004.

Line Mean plant

fresh weight (g)

Stem dry wt per plant (g)

Leaf dry wt per

plant (g)

Stevioside (%)

Rebaudioside A (%)

Iso-steviol (%)

Brazil 172 16 24 3.94 3.20 0.00 Herb Herbert 71 5 9 5.35 1.07 0.00 Richters 176 14 23 4.07 1.83 0.00 New Gippsland 180 20 18 6.53 2.55 0.00 Zhang Jingong 130 7 18 3.85 6.49 0.00 Sahin 151 13 20 6.47 2.57 0.00 T4 143 12 19 6.20 0.93 0.00 T2 187 15 25 7.61 1.40 0.00 94/8 150 12 22 7.72 1.72 0.00 83/27 158 9 21 5.72 2.81 0.00

Table 32. Fresh weight, stem dry weight, leaf dry weight and levels of stevioside, rebaudioside A and iso-steviol in extracts of ten stevia lines harvested on the 24 February 2004.

Line Mean plant

fresh weight (g)

Stem dry wt per plant (g)

Leaf dry wt per

plant (g)

Stevioside (%)

Rebaudioside A (%)

Iso-steviol (%)

Brazil 105 9 14 2.93 1.78 0.00 Herb Herbert 75 5 13 5.38 1.52 0.00 Richters 105 7 18 4.82 3.59 0.00 New Gippsland 105 6 16 4.77 1.98 0.00 Zhang Jingong 96 6 15 4.13 3.17 0.00 Sahin 102 6 15 4.36 2.00 0.00 T4 163 10 23 4.30 1.94 0.00 T2 112 6 16 4.62 1.87 0.00 94/8 166 10 22 5.31 1.53 0.00 83/27 212 14 29 4.85 1.91 0.00 Mean plant fresh weights and mean dry weights of stem and leaf were again variable between harvests and loss of leaf due to Botrytis may have contributed to this. Over the two harvest times, no significant differences between lines were found in the stevioside and rebaudioside A contents of leaf samples (Table 33).

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Table 33. Mean stevioside and rebaudioside A contents of leaf samples of lines, over two harvest times.

Line Stevioside (%) Rebaudioside A (%) Brazil 3.435 2.5 Herb Herbert 5.365 1.3 Richters 4.445 3.4 New Gippsland 5.65 2.3 Zhang Jingong 3.99 4.9 Sahin 5.415 2.6 T4 5.25 1.7 T2 6.115 1.8 94/8 6.515 1.8 83/27 5.285 2.4 Mean 5.147 2.293 lsd (P=0.05) ns ns

Poor seed germination initially caused delay in stevia investigations and seed quality was soon recognized as an issue. Freshly collected, mature seed generally had good germination characteristics. Vegetative propagation led to large variation in the time of flower initiation. While some lines flowered more readily than others, accurate comparisons of lines with regard to flowering time were not achieved. Leaf:stem ratios increased in early growth but appeared to reduce near the onset of flowering. Leaf diseases may have impacted on this change and identification of the time of maximum leaf yield is likely to be important in optimising yield of extract. Plant regeneration was rapid after early harvests of top-growth and the overall benefit of taking multiple harvests in one season should be investigated. The regeneration of stevia plants after the Tasmanian winter period was generally poor and improving this aspect should also be investigated, given the likely gains in yield and crop profitability, which may result from good plant stands in subsequent years. It is possible that breakage of senesced top-growth at the soil surface in autumn may have contributed to susceptibility to disease over winter. Autumn pruning and fungicide application may result in improvement in the wintering of plants. The absence of iso-steviol and the presence of significant levels of stevioside and rebaudioside A in all samples is a positive result. In test analyses, minor elution peaks were evident (elution times of approximately 11 and 13 minutes respectively) between stevioside and rebaudioside A and a significant unidentified peak was also present (elution time of approximately 19.5 minutes). Further work to establish the identity of these substances is necessary and confirmation may be available from the analyses of the complimentary samples sent to CQU for analysis.

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Stevia

42

8. Conclusions Astragalus was easily sown by standard seeding equipment and grew, flowered and set seed well under the climatic conditions of northern Tasmania. Plants wintered well after a first growing season but less well after the second season’s growth. Given the relatively mild winter climate in Tasmania in comparison those of some of the plant’s native habitat in China, improvements in the longevity of plants should be able to be improved with further study. As with some other medicinal herb species where the root is taken for extraction of actives, the efficient mechanical harvest of roots is likely to require innovation and adaptation of standard equipment. Levels of astragalosides in root samples from three crops of varying ages, of nine to 23 months at the beginning of the harvest period, were high compared with published data, which result is encouraging from the point of view that relatively young plant material was analysed in the current investigations and that concentrations of astragalosides in roots appeared to increase with crop age. In comparison, the age of plants in commercial harvests in China is usually more than five years old. This work has indicated that astragalus products may be produced locally and, while an extended period is needed for the analysis of growth, concentrations of actives and profitability, the indications that local production would be competitive are good. After a difficult period during the establishment phase of bilberry, field plantings in Tasmania have flowered and produced a few fruit in their third year of growth, which timing is consistent with its growth in native habitats. While insufficient fruit was obtained in this initial project to investigate extracts, market drivers appear to be strong. Further work is needed to determine the yield potential of harvested fruit and extract in the Tasmanian environment. Particularly the growth and flowering responses of bilberry to seasonal temperature and shading regimes need to be better determined. Variation among individual bilberry plants with respect to yield and quality of fruit extract is said to be high due to hybridism and attempts to identify highly productive individuals, which are suited to the Tasmanian climate, would be valuable to a future local industry. Significant developmental work is also required on ways of mechanizing the harvest of fruit, which are small and dispersed upon the plant. The preliminary evaluation of feverfew carried out in this project has found that the species grows and winters very well in the temperate climate of north- west Tasmania. Parthenolide yields have been found to vary considerably with season and plant management. While it may be possible to refine the timing of maximum concentration of parthenolide in feverfew by expanding such analysis into the winter period, total parthenolide yields are likely to be less than the levels found in summer regrowth. Further growth analyses, which consider the timing of trimming plants in relation to seasonal growth and the economic value of multiple harvests may be worthwhile. While the market for feverfew at the time of writing this report is well supplied, these encouraging results have convinced the industry stakeholder, BRA, to maintain feverfew plantings beyond the life of the current project. A current detraction to the development of stevia is the banning of the importation of stevia products by the European Union, its restriction in US and the possibility of its review in Japan, a major market, due to questions of safe use. For these reasons, BRA has decided not to pursue the development of this species at this time but to continue collaboration with the CQU project.

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Heptinstall, S., Awang, D.V., Dawson, B.A., Kindack, D., Knight, D.W. and May J. (1992). Parthenolide content and bioactivity of feverfew (Tanacetum parthenium (L.) Schultz-Bip, Estimation of commercial and authenticated feverfew products. J. Pharm. Pharmacol. May, 44, (5), p391-5. Huang, Y. and Guo, A. (1996). Investigation and production of type R-A steviosides. Journal of Plant Resources and Environment. 5, (4), p29-32. Hutapea, A. M., Toskulkao, C., Buddhasukh, D. Wilairat, P. and Glinsukon, T. (1997). Digestion of stevioside, a natural sweetener, by various digestive enzymes. Jour. Clinical Biochem. Nutrition. 23, p177-186. Ihalainen, M. and Pukkala, T. (2001). Modelling cowberry (Vaccinium vitis-idaea) and bilberry (V. myrtillus) yields from mineral soils and peatlands on the basis of visual field estimates. Silva Fennica. 35 (3), p329. Johnson, E.S., Kadam, N.P, Hylands, D.M. and Hylands, P.J. (1985). Efficacy of feverfew as prophylactic treatment of migraine. Br. Med. J. (Clin Res Ed). 291, (6495), p569-73. Kang, B.Y., Chung, S.W., Kim, T.S. (2001). Inhibition of interleukin-12 production in lipopolysaccharide-activated mouse macrophages by parthenolide, a predominant sesquiterpene lactone in Tanacetum parthenium: involvement of nuclear factor-kappaB. Immunol. Lett. 77(3, p:159-63.

Kinghorn, A.D., (2002). Stevia. The genus stevia. A. D. Kinghorn (ed.). Medicinal and aromatic plants – industrial profiles. Taylor and Francis. London and New York. Kolb, N., Herrera, J.L., Ferrega, D.J. and Uliana, R.F. (2001). Analysis of sweet diterpene glycosides from S rebaudiana – improved rapid HPLC method. J. Agric. and Food Chemistry. 49, (10), p4538-41. Lin, L.Z., He, X.G. and Lindermaier, M. (2000). Liquid chromatography-electrospray ionisation mass spectrometry study of the flavonoids of the roots of Astragalus mongholicus and A. membranaceus. J. chromatogr. A. 876, p87. Ma, X. Q., Shi, Q., Duan, J.A., Dong, T. T. X. and Tsim, K. W. K. (2002). Chemical analysis of Raix Astragali (Huangqi) in China: a comparison with its adulterants and seasonal variations. J. Agric. Food. Chem. 50, p4861.

Madhavi, D.L., Bomser, J., Smith, M.A.L .and Singletary, K. (1998). Isolation of bioactive constituents from Vaccinium myrtillus (bilberry) fruits and cell cultures. Plant Science 131, (1), p95 –103. Matsui, M., Sofuni, T. and Nohmi, T. (1996). Regionally-targeted mutagenesis by metabolically-activated steviol: DNA-sequence analysis of steviol-induced mutants of guanine phosphoribosyltransferase (gpt) gene of Salmonella typhimurium TM677. Mutagenesis 11, p565-572. Mittra, S., Datta, A., Singh, S.K., Singh, A. (2000). 5-Hydroxytryptamine-inhibiting property of Feverfew: role of parthenolide content. Acta Pharmacol. Sin 21, (12), p1106-14. Murashiga, T.and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Planta. 15, p473-497. Nakayama, K., Kasahara, D. and Yamamoto, F. (1986). Absorption, distribution, metabolism and excretion of stevioside in rats. Journ. Food Hygiene Soc. Japan. 27, p1-8. Nelson, M.H., Cobb, S.E., and Shelton, J. (2002). Variations in parthenolide content and daily dose of feverfew products. Am. J. Health Syst. Pharm. 59, (16), p1527-31. Nyman, N.A. and Kumpulainen, J.T. (2001). Determination of anthocyanidins in berries and red wine by high-performance liquid chromatography. Journal of Agricultural and Food Chemistry. 49 (9): p4183. Piela-Smith, T.H., Liu, X. (2001). Feverfew extracts and the sesquiterpene lactone parthenolide inhibit intercellular adhesion molecule-1 expression in human synovial fibroblasts. Cell Immunol. 209, (2), p89-96. Pietta, P., Simonetti, P. and Mauri, P. (1998). Antioxidant activity of selected medicinal plants. Journal of Agricultural and Food Chemistry. 46, (11), p4487- 4490. Rank, A. H. (1999). Stevia rebaudiana. A review. Final report of a project UCQ 16A to the Rural Industries Research and Development Corporation. Ritchie, J. C. 1956. Biological flora of the British Isles: Vaccinium myrtillus L. Journal of Ecology. 44(1): 290-298. Rogers, L. (1976). Effects of mast and berry crop failures on survival, growth, and reproductive success of black bears. Transactions, North American Wildlife Conference. 41, p431-8.

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Toda, S. and Shirataki, Y. (1999). Inhibitory effects of Astragali radix, a crude drug in oriental medicines, on lipid peroxidation and protein oxidative modification by copper. Journal of Ethnopharmacology. 68, p331-333. Toyoda, K., Matsui, H., Shoda, T., Uneyama, C.,Takada K. and Takahashi, M. (1997). Assessment of the carcinogenic activity of stevioside in F344 rats. Food Chem. Toxicol. 35, (6), p597-603. Van der Kloet, S. P. (1983). The taxonomy of Vaccinium and Cyanococcus: a summation. Canadian Journal of Botany. 61, (1) p256-66. Van der Kloet, S. P. (1988). The genus Vaccinium in North America. Publication 1828. Ottawa: Research Branch, Agriculture Canada. 201 p. Vanninen, I. Laakso, S. and Raatikainen, M. (1988). Geographical variation in the phenology and morphology of bilberry in Finland. ActaBot. Fennica. 136, p49-59.

Vogler, B.K., Pittler, M.H. and Ernst, E. (1998). Feverfew as a preventive treatment for migraine: a systematic review. Cephalalgia. 18, (10), p704-8. Wilkinson, J.A., Wahlquist, M.L. and Clark, J. (2002). New food and pharmaceutical products from agriculture. Papers from Outlook 2002. A report for the RIRDC. RIRDC Publication No. 02/05. February 2002. Xiao-hong et al., (2003). Supercritical carbon dioxide fluid extraction of the saponin in Anemone raddeana Regel. Hunei Daxue Xuebao, Ziran Kexueban, 25 (2), p156-9 (Abstract only of Chinese text). Zhang, ZiZhong. Liang, XinMiao. Zhang, Qing. and Lu, PeiZhang. (2001). Characterization and recognition of key components in Astragalus membranaceus. Acta Pharmaceutica Sinica 36 (7): p523. Zhou, K.S., Mancini, C. and Doria, G. (1990). Enhancement of the immune response in mice by Astragalus membranaceus extracts. Immunopharmacology. 20, p 225-234.

46

APP

Tasmanian Institute of Agricultural Research

Overview of the EU herbal remedies and nutritional

supplements market

7th December, 2001

EURO-LINK CONTACT Sally Chandler

Tel. + 39 02 7712 1124 Fax. + 39 02 7712 1130

sally.chandler@austrade.gov.au

Appendix 1

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THE CLIENT: The Tasmanian Institute of Agricultural Research (TIAR), at the University of Tasmania’s Burnie campus, is investigating the potential for the broadacre farming of four medicinal crops with Tasmanian company - Botanical Resources Australia (BRA). The four crops are:- Feverfew Tanacetum parthenium Bilberry Vaccinium myrtillus Stevia Stevia rebaudiana Astragalus Astragalus membranaceus Traditional Uses include:- Feverfew Migraine headaches, arthritis and painful menstruation. Bilberry Night blindness, hardening of arteries, eye cataracts, diahorrea, varicose

veins. Stevia Used as a sweetener. Astragalus Tonic for increasing energy levels, stimulating the immune system – also as a

diuretic, a vasodilator and treatment for respiratory infections. TIAR are seeking an overview of market requirements and potential for the four products together with an indication of market prices which could be obtained so that they and in turn BRA, can carry out a feasibility study in order to make decisions regarding continuing with production of the crops. OVERVIEW OF THE EU HERBAL REMEDIES AND NUTRITIONAL SUPPLEMENTS MARKET: General European Union issues for consumer-ready phyto medicines: Packaging Products entering the European Union (EU) must include instructions for use in all languages for the markets in which they will be sold. Some countries also have specific packaging laws in addition to the EU regulations that must be met by exporters to the EU. Labelling Labelling is strictly controlled in the EU and must provide consumers with precise information which answers six basic questions about the product: • What is it? • What is it used for? • What is it made of? • What does it cost? • How is it used? • When should it be used?

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Each European Union country is integrating the rulings of the EU into its national legislation. As much legislation regarding labelling is still in the developmental stage, EU labelling regulations and standards need to be carefully monitored. ESCOP: ESCOP was founded in June 1989 as an umbrella organisation representing national phytotherapy associations across Europe, especially in their discussions with European medicines regulators. Since 1996 it has been a company limited by guarantee in the United Kingdom. ESCOP has the following Aims and Objectives. Aims: To advance the scientific status of phytomedicines and to assist with the harmonisation of their regulatory status at the European level. Objectives: ♦ to develop a coordinated scientific framework to assess phytomedicines; ♦ to promote the acceptance of phytomedicines, especially within general medical practice; ♦ to support and initiate clinical and experimental research in phytotherapy; ♦ to improve and extend the international accumulation of scientific and practical knowledge in

the field of phytotherapy; ♦ to support all appropriate measures that will secure optimum protection for those who use

phytomedicines; ♦ to produce reference monographs on the therapeutic use of plant drugs; ♦ to further cooperation among national Associations of phytotherapy to advance these Aims

and Objectives. ESCOP achievements ♦ The publication in 1997 of 50 Monographs on the Medicinal Uses of Plant Drugs submitted

to regulatory authorities across Europe and accepted by the ad hoc Working Group on Herbal Medicinal Products of the European Agency for the Evaluation of Medicinal Products (EMEA) as providing the basis for proposed core-SPC’s for European decentralised marketing authorisations.

♦ An ongoing programme to complete further monographs. ♦ The co-ordination of the EU BIOMED research programme Determining European standards

for the safe and effective use of phytomedicines. Outcomes included, amongst others, the production of the ESCOP monographs, the establishment of an on-line reporting system for adverse reactions to herbal remedies (PhytoNET at http://www.escop.com), and the appointment of a Toxicology Panel.

♦ The publication of a professional newsletter, The European Phytojournal, available for free downloading on the Web at the PhytoNET address.

♦ The organisation of five International Symposia in Brussels, Milan, the Hague, and Cologne, with the latest in London in October 1998 under the title Phytomedicine and Consumer Protection.

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ESCOP Definition of Phytomedicines Phytomedicines, or herbal medicinal products, are medicinal products containing as active ingredients only plants, parts of plants or plant materials, or combinations thereof, whether in the crude or processed state. Notes on the Definition ♦ Medicinal products are defined in European Directive 65/65/EEC. ♦ The production of phytomedicines is formally determined in "Quality of Herbal

Remedies" in "The rules governing medicinal products in the European Community, Volume III, Guidelines on the quality, safety and efficacy of medicinal products for human use" (ISBN 92-825-9619).

♦ Plant materials include juices, gums, fixed oils, essential oils, and any other directly derived crude plant product. They do not include chemically defined isolated constituents, either alone or in combination with plant materials.

♦ Phytomedicines may contain excipients of plant or non-plant derivation. Useful Internet links

The most comprehensive information on EU Certifications for health and pharmaceutical products can be found on: http://pharmacos.eudra.org/

http://dg3.eudra.org/ is the website of the European Commission Pharmaceuticals and Cosmetics In http://dg3.eudra.org/eudralex/index.htm you can find all the Rules Governing Medicinal Products in the European Union

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MARKET INFORMATION: EUROPE

General Overview: The European market for natural remedies has grown and is growing rapidly as demand for natural, alternative remedies continues to increase. The enormous growth in the herbal medicine market can be attributed to a number of changes in the health sector. One primary factor is that due to the increased life expectancy in Europe and the increase in chronic diseases people are turning to herbal medicines out of dissatisfaction with conventional medicine’s inability to deal with these diseases. Many patients in fact refuse to take conventional medicine and consult their doctors for “natural” medicines. Other reasons for growth in the market are the: • continued promotion of the benefits of healthy living, preventative medicine, and self-

medication leading to greater public awareness of how supplements can benefit them; • EU population is becoming more middle-class and as a result of greater education and

knowledge of health issues, the middle class is more likely than their parents to seek health advice and use supplements;

• success of many products is due to their effectiveness in ameliorating age-related conditions and as the EU population is ageing, this will continue to fuel demand for many products.

All segments of the market are forecast to grow over the next few years, although growth rates will vary from segment to segment. Best prospects include: • Single vitamins: antioxidants are the most likely sales prospects in Europe. Anti-oxidants

such as beta-carotene, vitamin A, vitamin C and vitamin E have had a significant amount of media coverage highlighting their benefits in combating illnesses such as heart disease and cancer.

• Multivitamins: about 20 percent of the nutritional supplement market is held by multivitamins. Combined and special formulas of nutritional supplements for individual segments of the market are among the best prospects for the EU market as specific formulas can be targeted at specific market niches.

Whilst most herbal remedies are marketed as “Food supplements”, there appears to be a growing interest in the medical community for complimentary products. Due to the growing public demand, sales of herbal remedies are now achieved mainly through health food stores, high street chemists, mail order, supermarkets and via the Internet. Import tariffs European Union member countries apply the EU common external tariff to goods imported from non-EU countries. The Community Integrated Tariff (TARIC) system is based on the Customs Cooperation Council Nomenclature (CCCN), also referred to as the Harmonized System (HS). This system was introduced to provide a standard tariff classification regime for all products imported and exported throughout the world.

51

Import duty for herbal remedies from Australia to EU ranges from 0% to 4%. Import duties

are calculated on an ad valorem basis, i.e. expressed as a percentage of the value of the imported goods.

Taxes and Other Import Fees The following in addition to import duties are value added taxes (VAT) charged in the individual EU member countries. The VAT is based on the CIF value plus duty. Austria- 20% Belgium- 21% Denmark- 25% Finland- 22% France- 19.6% Germany- 16% Greece- 18% Ireland- 21% Italy- 20% Luxembourg- 15% Netherlands- 17.5% Portugal- 17% Spain- 16% Sweden- 25% United Kingdom-17.5% For further information on EU Commission Customs and Indirect Taxation: Director General Customs and Indirect Taxation (DG-XXI) Rue de la Loi 200 1049 Brussels Tel : (32-2) 296-1169; Fax: (32-2) 295-6501 Trade Fairs The following Trade Fair is probably the most important trade event of the sector and it may be of interest to TIAR and BRA: ♦ Biofach - World's organic trade show, Nuremberg, February 2002 (Interval 1 year)

Further information: http://www.biofach.de/d/index_2.html

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SELECTED MARKETS

GERMANY Overview of the market Germany is the world’s largest market for phytopharmaceutical products. German consumers are aware of most herbal products and tend to use these frequently. About 80% of doctors in Germany use herbal medicine regularly. General practitioners and pharmacists also promote the use of herbal products among their customers. The German population is advancing in age; every fifth German is now over 60 years old. In the year 2030, it is expected that it will be every third. Based on this development and the growing health awareness, demand for vitamins and minerals is also growing considerably. Traditionally, vitamins were only prescribed by physicians in cases of medical deficiencies, pregnancy and lactation, and to children. Recently, however, the German Ministry of Health has advised smokers and fast-food consumers to increase their intake of vitamins and minerals, and, consequently, calcium and magnesium, vitamins, mineral combinations, ginseng and St. John’s wort are becoming more popular. Large phyto-companies such as Schwabe, Finzelberg, Madaus and others have invested considerable time, financial resources and marketing to define and register monographies for most herbal extracts/products. German companies then buy these registrations together with a 10-year raw material supply – contract from large Phyto-companies, to formulate and manufacture their own products. Also the medical multinational companies are discovering the potential of this market sector and there is a trend of concentration of ownership (multinationals buying smaller companies specialised in herbal medicine or increased Joint Ventures). In Germany, about 40% of the cost of herbal medicines to consumers is reimbursed by Health Insurances Companies. Restrictions and Issues Most herbal extracts come under the phytopharmaceutical registration act in Germany. This registration process is similar to that of pharmaceuticals. Registrations can take very long and involve substantial financial resources. The possibility to import consumer-ready health supplements and herbal remedies to Germany depends on the contents of the products, which may be classified as either medicine or food supplements. This depends on the dosage and contents of the product, as well as package and leaflet design. Imported products are classified by customs upon arrival of the shipment. In cooperation with local health authorities (Gesundheitsbehoerden), custom authorities will categorise imports as either food or pharmaceuticals.

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Regulations and Regulating Authority There has been a long discussion about herbal medicine in Germany and within the European Economic Community (EEC). In Germany, this resulted in including specific regulations in its 1976 Medicines Act. The so-called Commission E was established by the Federal Board of Health to prepare monographs for herbal medicines. More than 230 positive monographs based upon data regarding the safety and efficacy were published in the German Federal Gazette “Bundesanzeiger”. These herbal medicines can be marketed according to the indications in the monographs. If the Health Insurances reimburse these kinds of indications, they do not differentiate between a chemical or herbal medicine. The German Medicines Act (AMG) as well as the Council Directive 65/65 European Economic Community (EEC) applies fully to herbal medicinal products (just as with chemical medicinal products). The European Court of Justice confirmed this in 1992. A marketing authorisation according to Article 4 of Council Directive 65/65 EEC granted by the competent authority is obligatory if herbal medicines are sold as finished medicinal products. The applicant must document quality, efficacy and safety of its product in compliance with CD 75/318 EEC. For further information regarding authorisation and registration of medicinal products please view: http://www.bfarm.de/gb_ver/drugs/#II\ In-market contacts and useful links Gesellschaft für Phytotherapie e.V. (GfP) attn. Prof. Dr. HD. Reuter Siebenebirgsalle 24 D-50939 Köln Germany Tel: +49 221 420 1915, Fax: +49 221 941 7020. http://www.phytotherapy.org/ (unfortunately this website is only in German language) UNITED KINGDOM Overview of the market In the UK, nutritional supplements are a sub-sector of the over-the-counter products (OTC) market, also referred to as self-medication aimed at generating health, not as illness remedies. This sub-sector includes three main areas: vitamins, minerals, and nutritional. Vitamin and mineral supplements account for around 7% of OTC sales in the UK. However, they have the largest advertising spend of all healthcare products, $40 million, about $5m more than analgesics. This sector spawns many new preparations each year, as companies try to keep market share. Private label is also important, with retailers like Boots, Superdrug, Sainsburys and Tesco all offering their own wide selections of vitamins and supplements.

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Distribution channels Pharmacies are the main distribution channel for nutritional supplements. About 70 percent of all OTC products sales are made through pharmacies, of which there are 11,000 in the United Kingdom. Boots the Chemists, Ltd. is the leading pharmacy chain, holding 34 percent of the total market. Around 800 of their stores have pharmacists trained in homoeopathic medicines. Another leading pharmacy chain is Lloyds, which owns the Holland and Barrett health food store chain. Holland & Barrett is the leading main street health food chain with around 350 stores. Independent pharmacies, however have seen a decline in market share, as the chains grow and the food multiples install in-store pharmacies. Health Food Stores are the second largest channel of distribution; however, the number of independent outlets (approximately 1,400) are declining with the increase of grocery multiples and chemists carrying health products and the growth of the Holland & Barrett health food store chain. Mainstream retail outlets such as grocery multiples are a rapidly-growing distribution channel. Supermarkets currently hold 16 percent of the OTC market after a growth of about 25 percent. In the United Kingdom there is a small group of grocery multiples, including Asda, Sainsbury, Tesco, and Safeway, with more than 1000 superstores between them. These superstores are aggressively seeking to expand their share of the nutritional supplements market. Many superstores now have a franchised pharmacy on the premises. Direct marketing and mail order of nutritional supplements is done by a relatively small portion of companies, such as Nature's Best. This method of distribution is expected to gain popularity over the next few years. There are also a number of companies that are selling supplements online, such as GNC (www.gnc.co.uk), the UK’s largest online store for vitamins and supplements. Import Regulations and restrictions Although the UK nutritional supplements market is relatively open to imports, market entry is not a simple process. The UK has stricter laws on what can and cannot be claimed in labelling and advertising of herbal remedies and nutritional supplements. Any product that makes a beneficial claim must go through the lengthy medicines approval process. If instead The Natural Way’s products are classified as food supplements, they will have to comply with UK food laws. These cover everything from approved ingredients, labelling standards and the need for an EU contact address. Totally prohibited by food law are claims (direct or implied) that a food is capable of "preventing, treating or curing any injury, ailment or adverse condition of the human mind or body," unless the supplement is licensed as a medicine under the Medicines Act of 1968. Nutritional supplements are exempt from the Nutritional Labelling Directive (90/496/EEC) which is aimed at food generally. Supplements in the UK are covered under the Health Food Manufacturers Association's (HFMA) voluntary Code of Advertising Practice.

55

Although this Code is voluntary, it is based on current regulations, and companies that do not follow it will consequently probably break the law. The HFMA offers fee-based advice to companies independently on labelling and advertising, and satisfying the requirements of the Code. Advice on labelling and promotional requirements for a given product will depend on the ingredients of the product, therefore a degree of disclosure of ingredients to authorized bodies will almost certainly be necessary. For further info please contact: Health Food Manufacturers Association (HFMA) 63, Hampton Court Way Thames Ditton Surrey KT7 0LT Contact: Maurice Hanssen Tel: +44 181 398 9888 Fax: +44 181 398 6906 Vitamin products, which contain herbals like ginseng and guarana, are usually classified as a food supplement, rather than a pharmaceutical product. For further information on duty rates and VAT please contact: HM Customs and Excise, Finchley Excise Berkeley House, 304 Regents Park Road London N3 2JY Tel: +44 20 7865 3100 Fax: +44 20 8346 9154 www.hmce.gov.uk SALLY CHANDLER, Market Entry Adviser, Austrade Euro-Link Centre, Milan – Italy. Tel: +39 02 7712 1124 Fax: +39 02 7712 1130 Email sally.chandler@austrade.gov.au This report was prepared in conjunction with : Stephanie McDonald, Business Development Manager, Austrade London Robert Bergmann, Business Development Manager, Austrade Frankfurt

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© Copyright Australian Trade Commission 2002

All rights reserved. The property and copyright in all material created in connection with this report shall vest in the Commission, and shall not be reproduced, without the prior approval of the Commission in writing.

Requests and inquiries concerning reproduction and rights should be addressed to

Manager, Legal and Risk Management, Australian Trade Commission, GPO Box 2386, Canberra, 2601. 56

EU Phytopharmaceuticals/OTC’s

TASMANIAN INSTITUTE OF AGRICULTURAL

RESEARCH

Europe

October 2002

Contact: Robert Bergmann Austrade Frankfurt Grueneburgweg 58-62 Frankfurt/Main Ph.: + (49 4254) 801980 Fax: + (49 4254) 801994 Email: robert.bergmann@austrade.gov.au

Appendix 2

EU Phytopharmaceuticals / OTC’s TIAR - Research

October 2002

Information or advice set out in this document is believed to be complete and correct at the time of publication. However, the Australian Trade Commission, its officers, employees, and agents disclaim any responsibility for loss or damage that may be suffered as a result of reliance on this information.

57

Contents

Page 1. Executive Summary 2-4 2. Market Size by Country 4-6 3. Market Size by Sector 7-9 4. Market Share 9-13 5. Market Forecasts 13-17 7. European Market News 18-28

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1. Executive Summary Confidence in Self-Medication Grows Western Europe saw sales of OTC healthcare products decrease by 6% in US dollar terms in 2000, with growth adversely affected by the continued weakness of the euro against the dollar. However, growth was positive in the vast majority of national markets in local currency constant value terms, as rising levels of health consciousness led consumers to take greater responsibility for their general wellbeing. The market also benefited from government-led initiatives, which encouraged consumers to self-medicate, due to the increasing financial burden of the ageing population on the health system. The ongoing liberalisation of distribution was accompanied by a marked increase in advertising activity, which was also instrumental in raising consumer awareness of OTC medicines in general. One significant factor affecting the OTC healthcare market in 2000 was a growing interest in products with natural ingredients, which are considered to be a safer as well as effective form of self-medication. Modest Growth in Germany Linked to Extension of “Negative List” Germany represented the largest market in Western Europe, with a 22% share of value sales in 2000. The shift towards self-medication was aided by the extension of the "Negative List", which comprises groups of drugs that are not reimbursed by Krankenkassen (semi-autonomous insurance agencies). However, value growth was constricted by the fact that a sizeable proportion of consumers chose to replace established brands with cheaper, generic products, which could be purchased in a wide range of outlets. Lack of Government Support in Italy Leads to Sluggish Sales In Italy, growth was hindered by the fact that there is a general lack of consumer awareness as to the uses and benefits of OTC products, a situation which arises from the fact that both the government and health professionals appear reluctant to promote self-medication. Despite the fact that it is now often cheaper to buy OTC products for minor ailments, many Italians continue to consult their GP (general practitioner – family doctor), a habit which harks back to the days when all medicines were completely free. Multinationals Dominant The OTC healthcare market in Western Europe remained highly fragmented in 2000, with a large number of multinational companies competing for share within what is a crowded marketplace. Overall, the market was characterised by a high number of mergers and acquisitions, as exemplified by the union of SmithKline Beecham and Glaxo Wellcome, as well as Warner-Lambert and Pfizer in 2000.

Roche ranked as the leading player in 2000, with a static 5% share of value sales, closely followed by Bayer and GlaxoSmithKline. The Boots Co registered a dynamic performance, following the acquisition of acne treatment brands Biactol and Clearasil from Procter & Gamble in autumn 2000.

EU Phytopharmaceuticals / OTC’s TIAR - Research

October 2002

Information or advice set out in this document is believed to be complete and correct at the time of publication. However, the Australian Trade Commission, its officers, employees, and agents disclaim any responsibility for loss or damage that may be suffered as a result of reliance on this information.

59

Distribution Shift Towards Grocery Outlets Chemists/pharmacies remained the most significant retail format for the distribution of OTC healthcare products across Western Europe in 2000, accounting for over 90% of total value sales in countries such as Belgium, France and Turkey. In general, the review period saw a shift away from pharmacies in favour of grocery outlets, as new government legislation resulted in further moves towards the liberalisation of distribution. Moderate Growth Predicted While a cautious approach to switching will continue to hamper value growth in Western Europe, particularly in countries such as Belgium, France and Italy, growing consumer interest in self-medication, fuelled by government-led initiatives designed to decrease the financial burden on the medical system, will drive the market forward between 2000 and 2005. Sales will continue to benefit from the liberalisation of distribution, while advertising will play an increasingly important role in driving growth in dynamic markets such Greece and Turkey, where sales will be boosted by rising standards of living, the establishment of better healthcare services and, in the case of the latter, increased levels of foreign investment. The "other OTC healthcare products" sector is expected to register the most dynamic rate of growth over the forecast period, underpinned by a continued boom in the popularity of niche products such as smoking cessation aids. 2. Market Size by Country The total value of the OTC healthcare market in Western Europe stood at US$17.2 billion in 2000, with value growth adversely affected by the continued weakness of the euro against the dollar. However, sales increased in local currency constant value terms, as greater levels of health consciousness, coupled with rising levels of affluence, led more people to take a pro-active stance regarding their general wellbeing. One general factor affecting sales of OTC products during the review period was a growing interest in self-medication, fuelled by government-led initiatives designed with the objective of decreasing the financial burden on the medical system. Demand for OTC healthcare products was also positively influenced by a rapidly ageing population, as manufacturers sought to increase the range of remedies targeted at this expanding consumer base. During the review period, self-treatment was further boosted by the widening availability of OTC products through grocery multiples, a trend which encouraged regular usage as more consumers purchased these remedies as part of their regular weekly shop. However, in countries such as Finland and Switzerland, market liberalisation placed pressure upon retail prices, which resulted in slow growth in real value terms.

EU Phytopharmaceuticals / OTC’s TIAR - Research

October 2002

Information or advice set out in this document is believed to be complete and correct at the time of publication. However, the Australian Trade Commission, its officers, employees, and agents disclaim any responsibility for loss or damage that may be suffered as a result of reliance on this information.

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Value growth was also attributable to the growing popularity of herbal remedies, such as echinacea, which as well as being renowned for their health-giving benefits, were attractive to consumers alienated by negative press given to chemical-based products. Moreover, product education by manufacturers combined with greater coverage of health issues in the print and broadcast media, led to a growing interest in preventative medicine, particularly vitamins and dietary supplements. Sales in Germany Boosted by Extension of “Negative List” With OTC healthcare sales totalling US$3.8 billion in 2000, Germany remained the most valuable market for self-medication in Western Europe, with a 22% share of value sales, down from 26% in 1996. Sales increased by 3% in 2000 in local currency constant value terms, underpinned by the extending of the "Negative List", which comprises groups of drugs that are not reimbursed by Krankenkassen. However, value growth was dampened by the fact that a sizeable proportion of consumers chose to replace established brands with cheaper, generic products, which are available through a wide range of outlets. One other significant factor affecting the OTC healthcare market in Germany in 2000 was continuing growth in interest in products with natural ingredients, in keeping with prevalent trends regarding the purity or "naturalness" of ingested products. Private Label Continues to Pose a Threat in the UK In the UK, the second largest national market in terms of OTC healthcare product sales, the market grew by a healthy 6% in local currency constant value terms in 2000, underpinned by a burgeoning consumer interest in self-medication, actively encouraged by a government intent upon reducing the financial burden on the NHS. This was particularly evident in the cough, cold and allergy remedies sector, as poor weather drove incidences of flu and other minor ailments to almost epidemic proportions. This prompted initiatives such as the NHSE (National Health Service Executive)’s Choose The Right Remedy campaign, urging cold sufferers to seek advice from pharmacists and purchase OTC products for coughs, colds and flu. Self-treatment was further encouraged by the widening availability of OTC products sold in grocery multiples, a large number of which now include in-store pharmacies. However, while increased distribution through grocery multiples benefited overall market growth, a concurrent increase in private label products posed mounting competition to branded medicines. Consequently, an increase in sales of products through grocery outlets was accompanied by rising advertising expenditure across all sectors, as manufacturers attempted to preserve their market share. Italy and Belgium Suffer from Lack of Government Support The country to experience the slowest rate of growth in local currency constant value terms in 2000 was Italy, where the habit of self-medication is still not as widespread as it is in other European countries. Despite the fact that it is now often cheaper to buy OTC products for minor ailments, many (especially older) Italians continue to consult their GP, a habit which harks back to the days when all medicines were completely free.

Growth is also hindered by the fact that there is a general lack of consumer awareness as to

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the uses and benefits of OTC products, a situation which stems from the fact that both the government and health professionals appear reluctant to promote self-medication. Other factors behind sluggish market growth in 2000 were low levels of product innovation, as well as Rx-to-OTC switch activity. During the review period, a similar situation was evident in Belgium, where a lack of enthusiasm on the part of the government, strong opposition from general practitioners and the concentrated structure of the retail network, prevented any significant growth in OTC sales over the review period.

Table 1 Value Sales of OTC Healthcare by Country 1996-2000

US$ million, current rsp

1996 1997 1998 1999 2000Germany 4,774.5 4,214.0 4,209.8 4,191.8 3,847.7UK 2,291.4 2,575.2 2,789.5 2,976.1 2,876.6Italy 2,468.4 2,471.2 2,629.7 2,599.4 2,298.4France 2,854.0 2,531.7 2,540.9 2,463.1 2,232.1Turkey 1,068.9 1,165.0 1,301.8 1,400.4 1,490.4Spain 1,077.4 975.8 1,008.3 1,020.1 970.9Switzerland 793.7 681.2 702.6 695.6 646.3Belgium 628.4 533.4 536.3 520.0 459.6Sweden 503.1 457.9 457.0 449.6 440.9Netherlands 451.9 405.4 421.7 433.1 406.0Austria 346.8 313.2 318.4 316.6 298.2Finland 341.6 302.1 290.9 290.0 265.9Norway 255.7 247.5 249.9 260.3 254.2Denmark 301.4 268.4 275.2 271.2 253.3Portugal 221.3 206.2 210.9 212.6 210.6Ireland 108.8 112.0 112.9 114.8 126.5Greece 92.9 97.6 97.5 101.7 84.3Other 72.9 67.6 68.1 67.9 64.8TOTAL 18,653.0 17,625.218,221.418,384.517,226.6

Source: Euromonitor

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3. MARKET SIZE BY SECTOR Table 2 Value Sales of OTC Healthcare by Sector 1996-2000

US$ million, current rsp

1996 1997 1998 1999 2000 1996/2000 CAGR

Cough, cold and allergy (hay fever) remedies 4,617.0 4,320.2 4,452.3 4,519.4 4,238.5 -2.1Vitamins and dietary supplements 4,127.8 3,870.2 4,004.4 4,024.5 3,729.8 -2.5Analgesics 3,674.7 3,515.4 3,663.9 3,708.2 3,486.9 -1.3Medicated skin care 2,239.7 2,163.4 2,250.4 2,275.7 2,134.4 -1.2Digestive remedies 2,261.9 2,101.2 2,121.8 2,094.6 1,917.0 -4.1Other OTC healthcare

1,731.9 1,654.9 1,728.7 1,762.2 1,720.1 -0.2

TOTAL 18,653.017,625.218,221.418,384.517,226.6 -2.0

Source: Euromonitor

Herbal versus Standard Standard products unsurprisingly continue to dominate the OTC healthcare market in Western Europe, accounting for between 85-97% of value sales in all four relevant sectors in 2000. In general, usage of herbal products increased over the review period, a trend which was attributable to a growing consumer interest in natural remedies, which are perceived to have fewer known side effects. Cough, Cold and Allergy (Hay Fever) Remedies Herbal/traditional remedies account for the highest proportion of total sales within the cough, cold and allergy (hay fever) remedies sector, where they are considered to be a safe and effective form of self-medication. In Germany, herbal products registered healthy growth over the review period, underpinned by the continued popularity of ivy-based expectorants, such as Prospan from Engelhard.

Meanwhile, in the UK, where the sector share accounted for by herbal remedies increased by 0.5 percentage points in 2000, growth stemmed from the established presence of manufacturers such as Nelsons and GR Lane, the latter of which supported its Olbas Oil natural decongestant range with a £1 million advertising campaign.

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As the consumer perception of herbal remedies became increasingly favourable, mainstream manufacturers expressed a growing interest in developing herbal ranges. One notable example was GlaxoSmithKline, which introduced an herbal cold remedy, Beecham’s Natural Relief Echinacea and Garlic into the UK market in October 1999. By contrast, growth was curtailed by the fact that in several countries, including Italy and Turkey, there are no registered herbal remedies within the cough, cold and allergy remedies sector.

Vitamins and Diatary Supplements The leading player in the vitamins and dietary supplements sector was Roche, which continued to enjoy a broad presence across a variety of subsectors, through its Supradyn, Sanatogen and Redoxon (Vitamin C) brands. In 2000, the company extended its Supradyn range of multivitamins with the launch of Supradyn 50+, a brand which capitalised upon the growing importance of the “grey pound”.

In the UK, one of Roche’s key markets, Sanatogen was relaunched in October 2000 in new blue and foil carton packaging, in a bid to accentuate brand identity amidst a plethora of diverse new vitamins and dietary supplements. The product range was streamlined to a core list of 29, and the brand was supported with a £5 million marketing campaign, to recover share lost to competitors such as Seven Seas. The second largest company in terms of sector sales was Merck, whose success was underpinned by its long-established Seven Seas brand, which has a strong presence in both Ireland and the UK. Like the majority of it competitors, Merck saw a slight gain in sector share, despite the ongoing fragmentation of the sector as increasing numbers of new products were launched. Ranking third in 2000 was Novartis, which owes its strength to its long-standing presence in the calcium supplements and fish oils sub-subsectors. Novartis has a particularly high profile in the Turkish market, where it held a 14% share in 2000 through its Ca-C-1000 and Calcium Sandoz Forte brands. In 2000, however, it gained half a percentage point in terms of its regional share largely due to the acquisition of the Hepar-SL forte brand in Germany in 2000.

Analgesics The analgesics sector was also dominated by standard products, with sales of herbal remedies generally limited to topical preparations. Growth was hampered by limited product availability, with many herbal analgesics proving ineffective compared to their chemical counterparts. Medicated Skin Care By contrast, herbal products accounted for a relatively modest 4% share of sector value within medicated skin care, with sales remaining negligible across much of the region. Growth was hampered by low product availability, with penetration highest in the antipruritics, medicated shampoos and haemorrhoid treatments subsectors. Digestive Remedies Within the digestive remedies sector, herbal OTC medicines accounted for 11% of value sales in 2000, with growth underpinned by the fact that they are perceived as offering a milder form of relief than standard drugs and, as such, they are less likely to upset the body.

The importance of herbal alternatives was particularly pronounced in the laxatives subsector,

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where natural ingredients such as Ispaghula husk and senna are widely used by both niche and mainstream manufacturers.

Table 3 Value Sales of Standard vs Herbal/Traditional OTC Healthcare by Sector: % Analysis 2000

% value

Standard Herbal/ Traditional

TOTAL

Analgesics 97.1 2.9 100.0Cough, cold and allergy (hay fever) remedies 85.4 14.6 100.0Digestive remedies 88.9 11.1 100.0Medicated skin care 96.4 3.6 100.0

Source: Euromonitor 4. MARKET SHARE The OTC healthcare market in Western Europe remained relatively fragmented in 2000, despite the high proportion of multinational companies present. Overall, the market witnessed a fair amount of movement towards the end of the review period, as there was a strong trend towards consolidation in the form of mergers and acquisitions, exemplified by the union of SmithKline Beecham and Glaxo Wellcome and Warner-Lambert and Pfizer in 2000. Roche ranked as the single most important player in 2000, its leading position underpinned by a strong presence in both the vitamins and dietary supplements and digestive remedies sectors. Despite substantial advertising investment, the company saw its market share remain static in 2000, as a variety of factors combined to pressure its share. The second ranked company in 2000 was Bayer, which continued to dominate the analgesics sector through its ubiquitous Bayer Aspirin brand. Despite the maturity of aspirin, Bayer maintained a static 4% share of value sales in 2000, following a strong performance from its Canesten antifungal brand. Following the merger of the UK-based multinationals Glaxo Wellcome and SmithKline Beecham in December 2000, the newly formed GlaxoSmithKline moved into the top three in Western Europe, helped by a strong presence in all six sectors. The company benefits from a portfolio of well-established international brands, including Panadol, Zantac and Zovirax. In 2000, the company initiated a number of product line extensions in an effort to underpin its OTC healthcare sales. One notable example was the Panadol analgesic brand, which now incorporates Panadol Soluble, Panadol Ultra, Panadol Extra, Panadol Junior and Panadol Night.

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One of the most dynamic players during 2000 was The Boots Co, which continued with its programme of expansion into Europe with the acquisition of acne treatment brands Biactol and Clearasil from Procter & Gamble in autumn 2000. Growth was also attributed to the solid performance of its heavily advertised Nurofen analgesic brand, which benefited from a switch from Rx-to-OTC status of certain formats in both France and Spain during 2000.

Private label sales remained static at just under 4% in 2000, with growth limited by the fact that in many countries, such as Spain, for example, strict regulation restricts the sale of most OTC products to pharmacies, and further regulation prevents the ownership of more than one pharmacy per person or company. The country where private label commanded the highest market share was the UK, where increased distribution through grocery multiples benefited market growth.

Table 4 Value Company Shares of OTC Healthcare 1999-2000

% value

1999 2000Roche Holding AG 5.0 5.0Bayer AG 4.3 4.4GlaxoSmithKline Plc – 4.3Novartis AG 3.9 4.2Pfizer Inc 0.9 3.8Boots Co Plc, The 2.3 2.8CH Boehringer Ingelheim Sohn 2.7 2.6Aventis SA 2.5 2.5Johnson & Johnson Inc 2.5 2.5Procter & Gamble Co, The 3.0 2.4Pharmacia Corp – 2.1American Home Products Corp 2.0 2.1Merck KGaA 1.5 1.5Beiersdorf AG 1.3 1.4Bristol-Myers Squibb Co 1.5 1.4Sanofi-Synthélabo SA 1.2 1.1Reckitt Benckiser Plc 1.0 1.0Pharmacia & Upjohn Inc 2.0 –SmithKline Beecham Plc 3.5 –Warner-Lambert Co 2.8 –Private label 3.7 3.7Others 52.4 51.2TOTAL 100.0 100.0

Table 5 Value Brand Shares of Analgesics 1999-2000

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% value

Brand Company 1,999.0 2,000.0Bayer Aspirin Bayer AG 14.6 14.3Nurofen Boots Co Plc, The 3.8 4.0Efferalgan Bristol-Myers Squibb Co 3.6 3.5Profenid Aventis SA 2.2 2.5Doliprane Aventis SA 2.5 2.4Voltaren Novartis AG 1.7 2.4Panadol GlaxoSmithKline Plc – 2.3Anacin American Home Products

Corp 2.0 2.1

Thomapyrin CH Boehringer Ingelheim Sohn

1.9 1.7

Ketofen Henkel KGaA 1.3 1.5Aspégic Sanofi-Synthélabo SA 1.4 1.3Calpol Pfizer Inc – 1.3Mobilat Sankyo Co Ltd 1.3 1.3Naprosyn Roche Holding AG 1.0 1.2Solpadeine GlaxoSmithKline Plc – 1.1Aspro Roche Holding AG 1.2 1.1ABC Beiersdorf AG 1.1 1.1Napren Elginkan 0.9 1.0Solpadeine SmithKline Beecham Plc 1.1 –Panadol SmithKline Beecham Plc 2.3 –Calpol Warner-Lambert Co 1.3 –Others 54.8 53.9TOTAL 100.0 100.0

Source: Euromonitor

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Table 6 Value Company Shares of Vitamins and Dietary Supplements 1999-2000

% value

1999 2000Roche Holding AG 11.3 11.6Merck KGaA 5.3 5.4Novartis AG 3.5 4.0GlaxoSmithKline Plc – 3.6American Home Products Corp 2.9 3.0Pharmacia Corp – 2.2MCM Klosterfrau GmbH 2.2 2.1Lichtwer Pharma AG 2.3 1.8CH Boehringer Ingelheim Sohn 1.5 1.6Beaufour-Ipsen SA 1.6 1.5Degussa-Hüls AG 1.5 1.4Herrn G Detlefssen AG & Co 1.1 1.1Groupe Juva Santé 1.0 1.0Arkopharma Group 1.0 1.0SmithKline Beecham Plc 3.5 –Pharmacia & Upjohn Inc 2.0 –Private label 7.8 8.1Others 51.5 50.6TOTAL 100.0 100.0

Source: Euromonitor

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5. MARKET FORECASTS By Country The OTC healthcare market will continue to expand at a moderate rate over the forecast period, with a predicted increase in constant value sales of 15% between 2000 and 2005. Growth will be boosted by an expected increase in self-medication, partly fuelled by government-led initiatives designed to decrease the financial burden on the medical system. Demand for OTC healthcare products will continue to be positively influenced by an ageing population, as manufacturers seek to increase the range of remedies targeted at this expanding consumer base. Another critical factor in boosting demand will be the growing popularity of herbal remedies, such as echinacea, as consumers show a greater interest in using products with so-called “natural” ingredients. During the forecast period, self-treatment will be further boosted by the widening availability of OTC products through grocery multiples, as more consumers purchase these remedies as part of their regular weekly shop. Concurrent with this trend, advertising will play an increasingly important role in driving growth, with OTC manufacturers having to invest to differentiate their brands from competitors in what will become an increasingly over-crowded marketplace. Continued Shift Towards Self-Medication in Germany and the UK In Germany, the most important country in the region in terms of sales of OTC healthcare products, the market is predicted to grow by 11% in real terms between 2000 and 2005, compared to constant value growth of 7% during the review period. Sales will be boosted by a growing consumer interest in self-medication, as increased levels of health consciousness, combined with greater time pressures, encourage consumers to take more responsibility for their own wellbeing, boosting sales of preventative medicine. The UK is predicted to experience above-average value growth over the forecast period, underpinned by a burgeoning interest in the convenience represented by self-medication, which will continue to be seen as a cost-effective, timesaving alternative to visiting a GP. Self-selection will be further encouraged by the increased prominence and availability of GSL medicines, with widening distribution through grocery multiples. Above-the-line advertising will grow in importance, particularly in the larger, maturing sectors such as analgesics and vitamins and dietary supplements, where manufacturers will attempt to offset market competition from cheaper, generic private label product ranges. Value growth will also be dependent on the continued introduction of products with added-value benefits, particularly within the cough, cold and allergy remedies and analgesics sectors, where there will be continued demand for high-strength, fast-acting products, such as Reckitt Benckiser’s Lemsip Max Strength and Whitehall Laboratories’ Anadin Ultra.

Dynamic Growth in Ireland, Greece and Turkey The country to experience the most dynamic performance over the forecast period will be Ireland, where a favourable economic climate, wider self-medication and an established government-led switching programme should further stimulate value growth. A similar pattern of sales will be seen in Turkey and Greece, where rising standards of living, a wider programme of health education and attempts to liberalise distribution network are expected to lead to increased sales of OTC healthcare products.

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Sluggish Sales in Belgium, France and Italy The country expected to witness the least growth over the forecast period is Belgium, where the development of the OTC healthcare market will continue to be restricted by the Government’s lack of interest in promoting self-medication. Sales will also be negatively affected by the reverse switch of codeine, which is available over the counter in neighbouring countries.

Meanwhile, in France, OTC medicines will continue to account for a relatively modest percentage of overall pharmaceutical sales, largely due to the well-established nature of the healthcare reimbursement system.

While the French government will certainly continue to promote the concept of self-medication, the traditional practice of the vast majority of French consumers to consult a doctor even for minor ailments is expected to constrain the potential positive impact of the switching and de-reimbursement policy on future OTC sales.

This trend is likely to be mirrored in Italy, where the reluctance of the government to encourage self-medication is unlikely to change significantly during the forecast period.

Table 7 Forecast Value Sales of OTC Healthcare by Country 2000-2005

US$ million, constant 2000 rsp

2000 2001 2002 2003 2004 2005Germany 3,847.7 3,921.4 3,993.7 4,072.7 4,161.9 4,258.8UK 2,876.6 3,018.9 3,149.0 3,269.1 3,371.1 3,466.2Italy 2,298.4 2,319.2 2,355.6 2,395.0 2,430.9 2,471.8France 2,232.1 2,248.1 2,267.8 2,287.8 2,305.8 2,327.2Turkey 1,490.4 1,580.4 1,678.1 1,783.0 1,895.6 2,015.6Spain 970.9 1,007.2 1,050.1 1,097.7 1,147.5 1,199.9Switzerland 646.3 666.1 683.9 701.6 719.0 736.6Netherlands 406.0 424.9 443.7 462.7 482.0 501.8Sweden 440.9 450.4 460.0 469.2 476.7 483.6Belgium 459.6 453.3 449.2 454.7 460.9 466.9Austria 298.2 306.7 315.1 322.7 331.1 340.0Norway 254.2 267.5 282.4 298.6 314.9 330.6Finland 265.9 269.8 274.4 279.6 285.5 292.2Denmark 253.3 258.9 266.5 274.3 282.3 290.1Portugal 210.6 216.9 223.7 231.2 239.4 248.2Ireland 126.5 134.2 143.4 153.4 164.5 176.7Greece 84.3 89.5 95.2 101.4 108.1 115.5Other 64.8 67.4 70.4 73.9 77.5 81.9TOTAL 17,226.617,700.918,202.218,728.719,254.519,803.7

Source: Euromonitor

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By Sector Vitamins and Dietary Supplements The vitamins and dietary supplements sector will witness steady growth over the forecast period, albeit at a slightly lower rate than the market as a whole. Sales will be boosted by a growing consumer awareness of the importance of preventative healthcare, as rising levels of media coverage, combined with busier lifestyles, encourage consumers to rely on such products in order to maintain a balanced diet and to ward off minor ailments. An expected surge in consumer interest for natural or herbal products such as ginseng, ginkgo biloba and echinacea, will continue to drive growth over the forecast period, fuelled by high-profile celebrity use of products mentioned in newspapers and magazines. In general, single vitamins will continue to lose ground to multivitamins, as the market for the former grows increasingly saturated. In countries such as Germany and the UK, private label growth is expected to escalate, underpinned by wider distribution of vitamins and dietary supplements through grocery multiples. In order to counter a potential decline in sales, particularly in the highly lucrative multivitamins subsector, manufacturers will continue to target specific consumer groups, including children, teenagers, pregnant women and the over 50s.

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Table 8 Forecast Value Sales of OTC Healthcare by Sector 2000-2005

US$ million, constant 2000 rsp

2000 2001 2002 2003 2004 2005 2000-2005

CAGRCough, cold and allergy (hay fever) remedies 4,238.5 4,352.2 4,460.1 4,565.4 4,669.5 4,771.1 2.4Vitamins and dietary supplements

3,729.8 3,824.6 3,923.1 4,025.8 4,129.4 4,243.8 2.6

Analgesics 3,486.9 3,566.7 3,664.4 3,777.5 3,883.9 3,995.7 2.8Medicated skin care

2,134.4 2,206.4 2,282.3 2,360.7 2,438.4 2,519.1 3.4

Other OTC healthcare 1,720.1 1,805.7 1,892.6 1,981.9 2,076.5 2,173.8 4.8Digestive remedies

1,917.0 1,945.3 1,979.7 2,017.3 2,056.8 2,100.1 1.8

TOTAL 17,226.617,700.918,202.218,728.719,254.519,803.7 2.8

Source: Euromonitor

Copyright Euromonitor 2001

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6. European market news Herbal Medicines Market Fails to Grow Figures released by the European Herbal Practitioners' Association (EHPA) show that the European market for over-the-counter (OTC) herbal drugs, which account for some 25% of the total OTC market, has experienced little growth during the past five years. During 2001 the herbal OTC sector reached US$2.8bn in terms of manufacturers' prices, with Germany and France controlling most of the sales with 39% and 29% of the total, respectively. Italy accounted for 7% of the herbal OTC market, followed by Poland and the UK with 6% each, and Spain with 4%. Although OTC drugs have become more popular as a result of higher public awareness about well-being and self-medication advances, herbal remedies remain a less attractive proposition than vitamins and similar products. © 2002 Dow Jones Reuters Business Interactive LLC (trading as Factiva). All rights reserved. Copyright 2002, World Markets Research Centre Limited. All Rights Reserved Stagnation on European over-the-counter herbal medicine market. According to EuropeanHerbals, the European market for OTC herbal drugs was worth $2.8 bn (manufacturer prices). Herbal medicines account for about a quarter of the European OTC market. With the exception of a few segments, the herbal market has had zero growth for a period of 5 years. A pie chart gives a breakdown of the market by country: Germany 39%, France 29%, Italy 7%, Poland 6%, UK 6%, Spain 4% and other 9%. Some of the smaller markets such as Spain have shown healthy growth. On the German market, sales of some osteomuscular treatments increased by 15%. Gynaecologicals such as Remifermin had 10% growth. © 2002 Dow Jones Reuters Business Interactive LLC (trading as Factiva). All rights reserved. (c) 2002 Elsevier Engineering Information www.ei.org NUTRACEUTICALS INTERNATIONAL 1st July 2002 EUROPEAN FOOD SUPPLEMENTS MARKET WORTH $2.66 BILLION, UP 7.7% IN 2001. There really is no industry view as to what constitutes the food supplements sector, according to Joachim Bug, general manager, R&D and operations at Merck KGaA, speaking at the 38th annual meeting of the Association of the European Self-Medication Industry (AESGP), which was held last month in Dublin, Ireland.

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However, he estimated that the sector in western Europe achieved sales of nearly $2.66 billion for 2001, and had grown 7.7% over the previous year, a level which he did not think was "very encouraging." Quoting figures from Nicholas Hall & Co, Dr Bug noted that the largest segment of the European food supplements market is vitamins, with sales last year of $917.4 million. This is followed by herbals and natural supplements, with turnover of $665.9 million, minerals at $600.5 million, "other" vitamins and minerals at $282.5 million and tonics with $193.4 million in sales. Asking the question, why do people demand food supplements, Dr Bug suggested that there are many answers to this. Many people want to compensate their bodies for their hectic lifestyles, and the same lies true for those who, for this or for other reasons, see malnutrition problems, he noted. Moreover, people just want to feel well. There is also the aging population, with dreams of eternal youth and a rising health consciousness. Additionally, scientific findings are providing justification for the use of supplements. Turning to regulation of the sector, Dr Bug said there is a need for harmonization of the rules, pointing out that the European Union's member states have diverse rules on permitted ingredients and dosages, as well as labeling, and he sees no reason for this. Need for pragmatism from regulators. Commenting on the proposed EU food supplements directive (NIs passim), he said great steps have been made but asked the regulators to take a more pragmatic approach, for example on minimum and maximum doses, pointing out that people in the UK, where high doses are permitted, are not dying as a result of these. However, Dr Bug said, what has been done with the directive is to regulate the past, and asked, how can we regulate the future? Importantly, he added, let us create the future; no real innovation is possible at present in Europe, and a framework needs to be created for this to happen. Referring to the proposed regulation on claims (see page 5), he said the European supplements sector has to support the requirement for adequate scientific substantiation relating to the level of label claims "as we have to be a reliable and trusted industry...otherwise this market will lose credibility as it did in the USA.". Opening the conference, AESGP president, Alessandro Banchi, noted that food supplements have become an important part of the association's activities. Pointing out that the directive on food supplements is now finally agreed after two years of intensive political debate, he said: "we will now have to monitor with great attention the numerous follow-up activities which will, at the end of the day, be decisive for what the directive will really mean in the market.". He went on to say that the AESGP is "awaiting with interest" the European Commission's announcement of proposals for food fortification and, in particular, for claims allowed for food products. On this, he observed, "there have been animated debates in the area of health and disease-risk reduction claims.".

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Addressing the question of herbal medicinal products, incoming AESGP president Albert Esteve welcomed the setting-up of an independent committee for these products within the European Medicines Evaluation Agency and said he was hopeful that this body will have the full competence to deal with all issues around herbals as soon as they come up at the level of the EMEA. Additionally, "the proposed legislation on traditional herbal medicines will hopefully create an adequate legal basis for this category of products and make a clear distinction between herbal medicinal products of traditional and of well-established use, " Dr Esteve concluded. Deboyser gives perspectives on food supplements. Patrick Deboyser, head of foodstuffs, at the legislation and scientific and technical aspects unit of the European Commission's Directorate General Health and Consumer Protection, confirmed that the food supplements directive should be published shortly, and noted that the Commission will set both the maximum and minimum amounts for permitted nutrients, but said it was undecided whether to do so in two steps or all at once. In either case, it expects to finish setting the levels for all of the nutrients by end-2003. Mr Deboyser said the Commission has noted several specific issues that remain to be resolved, including comparative claims among different foods, whether certain foods should be prohibited from health claims (eg wine), who has the burden of proof and what is its standard. He added that the inclination now is to permit claims regarding cognitive functions but prohibit psychological and behavioral functional claims. 58 NUTD © 2002 Dialog NUTRACEUTICALS INTERNATIONAL 1st February 2002 HERBALS DIRECTIVE TURNING THE SCREWS ON UNLICENSED PRODUCTS? Efforts to find a new regulatory home for herbal remedies under European Union drug law have gained momentum as the European Commission adopted its proposed directive on traditional herbal medicines. In the unlicensed herbals sector, however, there were lingering concerns about the latest draft of the proposals signed off by the Commission on January 17. Not only do they leave the gate closed to a number of marketed herbal products, it is argued, they will also encourage regulators to take a tougher line on herbs sold under food legislation or other fall-back options. This comes at a time when further legislative measures are set to narrow the scope for incorporating herbs into foods across Europe. Manufacturers of licensed herbal medicines, represented by the Association of the European Self-Medication Industry (AESGP), were far happier with the drift of the proposed directive, although not without qualms about its scope and certain ambiguities in the text. Provided "some critical details" were resolved, the proposals should "clarify the legal basis for many herbal products in the EU, " according to the AESGP's director general, Hubertus Cranz.

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The proposal for a directive on herbal medicinal products offers a third tier of registration for herbals, in addition to the standard drug approval route and the "well-established medicinal use" provisions originally published as Directive 1999/83/EC. These latter allow bibliographic data to substitute for the results of safety and efficacy testing where products meet the 10-year established-use criteria. The prompt moves to add a further layer to these options acknowledged that Directive 1999/83/EC would still leave many herbal products out in the cold. Registration waives efficacy requirement. At the core of the new proposals, adopted by the Commission as amendments to the consolidated Directive 2001/ 83/EC, is a simplified registration procedure that waives efficacy and, to a lesser extent, safety requirements where there is sufficient evidence from long-term traditional use to support approval. The qualifying period is 30 years' medicinal use within the EU, although experience in other territories may count if the product has been on the EU market for at least 15 years. Quality and manufacturing requirements, however, remain the same as for medicines taking the conventional approval route. The trade-off for concessions on safety and efficacy is that the potential indications, dosage and formulations of traditional herbal medicines will be limited. Labeling and advertising will have to carry the rider that the product is for traditional use and its efficacy has not been clinically proven. Moreover, traditional herbal medicines will not be eligible for mutual recognition. The wide discrepancies in the regulatory treatment of herbal products identified by the AESGP in its 1998 report for the Commission are seen as ruling out full harmonization for the time being. In assessing applications for herbal medicinal products, however, national regulators will have to "take due account of registrations or authorizations granted by another member state.". At the same time, the proposed directive raises the profile of herbal medicines by establishing a Committee for Herbal Medicinal Products at the European Medicines Evaluation Agency. One of the committee's main tasks will be to draw up herbal monographs as a blueprint for assessing applications under both the well-established use and traditional-use provisions. Once these monographs are adopted, traditional-use applications must be based upon them. The committee will set up a list of eligible herbal substances, together with indications, strengths, routes of administration etc, that will enable applicants to skirt requirements such as bibliographic and expert evidence on safety or 30 years' use. Impact on industry acknowledged. The impact assessment accompanying the proposed directive notes that it will affect "certain companies belonging either to the pharmaceutical or to the food industry, " as the aim is to permit "the marketing as medicinal products of products that so far do not have a defined status." The nervousness about the proposals in the unlicensed sector relates precisely to those markets that have allowed herbals to fall into the margins. Countries such as the Netherlands, Belgium and the UK have coped with the rising tide of new herbal products in Europe (particularly in the wake of the US Dietary Supplement Health and Education Act) by clearing space for them under food law or, in the UK's case, stretching a loophole in its drug legislation.

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The fear is now that the traditional-use provisions will justify a less indulgent attitude to herbs in these categories. Section 12 of the UK Medicines Act, which permits the unlicensed sale of dried or crushed herbs without brand names or claims, is regarded as particularly vulnerable in this respect. A highly lax interpretation of the exemption has provided an opening for numerous herbal products such as St John's wort, kava kava and echinacea. In parallel, the opportunities for marketing herbs in combination (eg with vitamins and minerals) as foods have contracted as the UK regulators apply the "medicinal by function" criterion with increasing stringency. Industry was recently assured that Section 12 would not be revoked in light of the proposed European directive. But the Medicines Control Agency has tried to jettison the exemption before (in 1993, unsuccessfully) and the indications are that, with the traditional-use amendments in place, Section 12 would be applied to the letter. This would make it virtually impossible to exploit the exemption on any meaningful commercial or industrial scale. There is similar unease about the liberal accommodations for herbs under food law in the Netherlands and Belgium. As one observer notes, these products have only survived on being "not illegal" rather than specifically lawful. The vacuum could be filled when the proposed EU directive on food supplements comes into play, as its definition of supplements has been expanded from vitamins and minerals to other ingredients with a "nutritional or physiological effect" - which would include herbal extracts. But this would only happen in the second phase of implementation and, by that time, it is suggested, other legislative measures in the pipeline may have scoured the EU food category of much of its herbal content. One estimate of the time taken to include herbals in this directive, coming out of the Commission, is 10 years. In the meantime, the unlicensed herbals sector wants to make sure its products are not locked into the traditional medicines channel, especially as the proposed directive throws up significant barriers to entry. Referring as it does to products, rather than ingredients or preparations, the 30-year rule may prove beyond the means of herbal remedies that have changed composition (eg from powders to standardized extracts) over the years, or indications from country to country (NI vol 6, no 11). Furthermore, their roots may only go as far back as the herbals boom of the late 1980s and 1990s. The proposed directive also makes clear that if a herbal medicine can be authorized under conventional drug approval criteria or the established-use provisions, then it should be. Coupled with the obligation to take other EU registrations into account (which, in countries such as Germany, would mostly be full medicinal approval), this raises the question of how many products would actually be admitted to the traditional-use stable. Drug- level requirements for manufacturing and quality assurance present another hurdle for the unlicensed sector, especially if products are being imported from the USA or other non-European markets. Coming from the pharmaceutical side of the fence, the AESGP said it was pleased the proposals had sought to confirm the distinction between traditional and well-established-use products. This has been a key consideration for the over-the-counter medicines industry, which was worried about the new provisions undercutting the status of herbal medicinal products with a sound scientific basis for established use (NI vol 6, no 7).

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Similarly, the association welcomed the clearly-defined mandate for the CHMP to deal with herbal medicines fulfilling both well-established and traditional-use criteria. Some question marks remained over the reference list of herbal substances, though, such as whether it would be comprehensive enough or whether it might weaken established-use credentials at national level. While the AESGP said it would have preferred the directive to take in the full range of traditional medicines, including combinations with ingredients other than herbs, most of its remaining concerns revolve around points of clarification. For example, the requirement for efficacy to be "plausible" on the basis of long-term use may be interpreted too stringently in some markets, it believes. 69 NUTD © 2002 Dialog MARKETING WEEK 16th August 2001 UK GETS THE URGE TO HERBAL Consumers in the UK are increasingly turning to "natural" remedies. Consumers consider them to be healthier and safer than synthetic or standard medicines. Euromonitor's latest research on the herbal remedies market shows that between 1996 and 2000 the UK market more than doubled, and is now worth #157m. This growth has been fuelled by a rising demand for alternative medicines, alongside increased product development and marketing activities. Euromonitor research shows that a higher profile in television and press advertising has influenced consumers' attitudes towards herbal remedies and has led to a greater acceptance of them as an alternative to standard medicines. Advertising campaigns for products such as GR Lane's Olbas Oil (costing #1m) and Beecham's Natural Relief Echinacea and Garlic (costing #1.6m) have helped to boost their sales growth. Homeopathic remedies are the leading sub-sector in the UK natural medicines market: in 2000, they accounted for 19 per cent of the natural medicines market. In recent years, homeopathy has benefited from a fashionable image as an exotic - and effective - alternative to over-the-counter (OTC) medicines. The widespread availability of an extensive range of homeopathic products in most leading health food shops and a large number of chemists/pharmacies has also aided sales growth. Natural cough and cold remedies have also experienced buoyant growth in recent years and by 2000 accounted for 12 per cent of the natural medicines market. Growth in this area has been stimulated by the entry of mainstream manufacturers and increased marketing activity. Although natural cough and cold products enjoyed a dynamic growth in 2000, they have had to compete against products such as Echinacea, which taken daily as a dietary supplement is said to boost the immune system. Euromonitor's study also reveals that natural remedies have fuelled sales in several OTC healthcare sectors. Growth has been most pronounced in the vitamins and dietary supplements

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market and by 2000, herbal and traditional medicines made up nine per cent of this #417m market. Increased consumer awareness of their distinct and individual health benefits has stimulated dynamic sales growth of Echinacea and St John's wort. The supplements sector has also seen expansion, with the novelty of exotic or unusual products such as kava-kava, green-lipped mussels and isoflavones driving consumer interest. The increased availability of products through mainstream retail channels has been another key driving force for the growth in sales of herbal cures. The UK has one of the most developed self-medication markets in Europe and consumers are taking greater responsibility for their personal health. As a result, retailers are stocking a wider range of healthcare products and providing advice on the benefits of herbal remedies as alternatives to OTC medicines. According to Euromonitor, health-food stores such as Holland & Barrett and Fresh & Wild are the most popular choices for consumers new to natural medicines because of their wide product range and knowledgeable staff. Last year, the health-food shops sector accounted for 52 per cent of the natural medicines' market and are the leading specialists in natural health products. However, increasing competition has arisen from grocery and pharmacy multiples, which have introduced more herbal products and own-label ranges. Last year, grocery outlets were particularly dynamic and their share of the natural medicines' market stood at nearly 16 per cent, representing a growth of more than five per cent since 1996. Chemists and pharmacies are also gaining market share. In 2000 they accounted for 22 per cent of the herbal remedies market, a rise of three per cent since 1996. Pharmacies have proven popular among consumers as a reliable source of information about using natural remedies as a replacement for standard OTC medicines. The variety of natural remedies has created a need for pharmacists to offer qualified advice on the benefits of herbal alternatives. This is in contrast to the focus on self-selection and brand awareness that characterises conventional OTC distribution. Euromonitor predicts that the UK natural medicines market will grow in value by 36 per cent this year. By 2003, sales of natural medicines in the UK are forecast to nearly double to #307m. Future growth will be driven by herbal equivalents of OTC products. In the near future, positive growth is expected from ginkgo, ginseng and Echinacea supplements, although more unusual supplements, such as saw palmetto, soy and kava-kava, will enjoy faster growth. Efforts on the part of manufacturers and retailers to become knowledgeable about healthy living will be important in driving sales. Chemists and health-food stores will continue to benefit because of their comprehensive range of products as well as their reputation for offering a high level of service. Copyright: Centaur Communications Ltd. and licensors © 2002 Dialog Western Europe market size for herbal products, expressed as sales in US dollars for each of the top seven countries or regions and "others" for 1999 and forecast for 2002

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Herbals and botanicals have been used for-centuries as remedies for a variety of ailments. The growing interest in alternative medicine and 'natural' alternatives to traditional medicines has, however, led to a dramatic growth in the herbals market over the last decade. FIONA ANGUS, market intelligence officer of the Leatherhead Food Research Association (LFRA) reports Herbal medicines and remedies remain the main application for herbals, but herbals have also been used in drinks for many years and are increasingly finding application in a variety of foods being promoted on a health platform. THE MARKET The global herbals market is now estimated to be worth in excess of $19Bn and is forecast to exceed $23Bn by 2002. Europe dominates the total market with a share of around $7Bn, followed by Asia and North America. Recent growth in the European and Asian market, has been steady but the American market has shown the most rapid growth, increasing from $1.5Bn in 1996 to $4Bn in 1999 and forecast to reach in excess of $6Bn by 2002 (Table I). The Western European market is dominated by Germany which accounts for around half of sales. Germany has a long history of use of herbal medicines, with many available on prescription. Market growth for herbals has slowed in Germany in recent years, however, as a result of a downturn in the economic situation and a national attempt to reduce health costs but future growth is expected from an increase in retail sales of herbal products. The French market has been experiencing slow/static growth; most growth in Europe is currently coming from the UK and Scandinavian markets. The UK market is currently estimated to be worth around $0.7Bn and is forecast to reach $0.8Bn by 2002 (Table Il). HERBALS AND BOTANICALS WITH HEALTH BENEFITS The European market for herbals and botanicals is dominated by sales of 'medicinal herbs'. Gingko biloba is the leading product in Europe, largely as a result of it being a popular prescription item in France and Germany. It is claimed to have hypotensive properties as well as beneficial effects on mental performance. St John's Wort is the next strongest market and it is gaining market share. Known as 'Nature's Prozac', St John Wort is used as a treatment for mild depression and largely used in the supplement industry, although its use in food and drinks products is increasingly evident. Other popular herbals and botanicals include valerian, which is thought to have sedative, relaxant and hypotensive properties. Ginseng is also important, claimed to have benefits for mental and physical performance and as an antistress agent. Guarana is becoming increasingly evident, used in supplements and in the soft drinks industry, largely as a stimulant added to energy drinks. Echinacea is also growing in popularity, taken largely for the prophylaxis of colds and influenza and as an immunostimulant. Green tea is beginning to gain popularity outside Japan and is likely to find increasing application in the food industry in the future. Claimed to have a variety of healthy benefits from anti-cancer

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to antibacterial properties, green tea has to date been used in a range of tea beverages and fruit drinks. HERBAL BEVERAGES Early Celtic beer contained herbs such as cumin, yarrow, burdock and nettle in its formulation, and mead was flavoured with fennel, cumin, rosemary and thyme. The popularity of these herbal-flavoured drinks continued until the second world war, when restrictions on the soft drinks industry led to their decline. Despite this, however, some soft drinks continued to contain herbs even though their presence was not promoted. Vimto and Dandelion & Burdock, for example, both contain herbal extracts. Over the last 10 years or so, there has been somewhat of a resurgence in herbal soft drinks, and we are also increasingly seeing the addition of herbal ingredients to a wider range of foods. The UK herbal soft drinks market is now estimated at UKPd65M, with products such as Aqua Libre and Ame being important. Elderflower cordial has also become popular and is sold as a drink to maintain well being. more recently, a range of herbal extracts is being added to soft drinks. Indigo, launched by Vimto Soft Drinks in 1996, is a sparkling energy drink containing wolfberry, schizandra and guarana extracts. Lipovitan is also increasing its share of the UK energy drinks market. This product from Taisho Pharmaceuticals and containing ginseng and royal jelly extract first appeared in the UK in 1996. The range has now been extended with Lipovitan ACE, containing antioxidant vitamins. Last year, SouthBeach Beverage company in the United States launched the SoBe range of herbal soft drinks in the UK. It is available in a number of varieties, Lizard Lightning contains ginseng, wolfberry and guarana; Lizard Fuel contain astralaus, ginseng and yerba mate; and Energy contains creatine, ginseng and guarana. Interestingly, we are also now seeing the emergence of herbals in the UK alcoholic drinks sector. Highland Distilleries introduced Grouse Rush last year, a pre-mixed spirit containing whisky and Siberian ginseng. TABLE 9: EUROPEAN HERBALS MARKET, BY VALUE, 1999-2002(f) Country 1999 ($Bn) 2002(f) ($Bn) Germany 2.2 2.5

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France 1.8 1.9 Italy 0.8 1.1 United Kingdom 0.7 0.8 Scandinavia 0.3 0.4 Netherlands 0.1 0.2 Spain 0.3 0.4 Others 0.4 0.5 Total 6.6 7.8 f = forecast Source: PhytoPharm Consulting, Berlin TABLE 10: GLOBAL HERBALS MARKET, BY VALUE, 1999-2002(f) Country/Region 1999 ($Bn) 2002(f) ($Bn) Western Europe 6.70 7.93 Eastern Europe 0.33 0.41 Asia 5.10 6.25 North America 4.00 4.73 Japan 2.20 2.67 Australasia 0.12 0.17 Africa and Middle East 0.12 0.14 Latin America 0.56 0.70 Rest of world 0.27 0.30 Total 19.40 23.30 f = forecast Source: PhytoPharm Consulting, Berlin In addition, herbal teas have been showing strong growth in recent years and the market is now estimated to be worth in excess of UKPd26M. Historically many of the more popular products have been fruit-based lines, marketed as caffeine-free alternatives to ordinary tea, but a variety of herbals and botanicals, including camomile, nettle and dandelion, is now being used in tea products.

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Ginseng is also being included in some teas that are being marketed on a health platform. A recent example includes Ginseng and Vanilla tea from R Twining & Co, containing liquorice root, hibiscus and ginseng. Developments in herbal-enriched foods have been slower to materialise, but new products are beginning to appear on the European market. Back in 1993, Rio Trading launched Buzz Bar and Buzz Gum onto the UK market; both products contain guarana. In 1997, Muller introduced what it claimed to be the first probiotic yoghurt to include herbal ingredients -- ProCult and Gesunheitskrauter, containing a herbal balm. In the United States, there are also savoury snacks containing herbals available on the market. Robert's American Gourmet produces a number of herbal-enriched lines, including Power Puffs, corn snacks with honey, ginseng and bee pollen. Worldwide there are a number important suppliers of herbals and botanicals. Many of them originate in the US such as Hauser Inc, for example, said to be the leading US supplier of herbal extracts and botanical raw materials, and Triarco Industries. There are some major players in Europe, however, who play a vital role in the global supply system. The Martin Bauer Group in Germany is thought to be the world's leading supplier of herbal materials to the tea, herb and extract producers. In 1999, the Company purchased P. Mueggenburg, its primary competitor in Europe, resulting in a combined revenue for the group of in excess of $400M Martin Bauer also owns a variety of other companies in the herbal area, including Plantextrakt and Finzelberg. The Company has also recently established a joint venture with American Ingredients to improve its share of the US market. Other important European players from Germany include Henkel, Schwabe and SKW Trostburg. Indena in Italy is, however, probably the next most important after Martin Bauer. The company has extraction facilities in France and recently bought Yew Tree Pharmaceuticals in the UK which it has renamed Indena UK. Indena and Schwabe dominate the supply of ginkgo; Martin Bauer does not supply ginkgo but dominates in St Johns Wort and echinacea. In the UK, herbals are now available from many of the major flavour companies, including Pointings and Quest. There are also some other important specialist suppliers in the UK, including William Ransom & Son and Bioforce (UK). EXPANSION CONTINUES In conclusion, the herbals market has enjoyed significant growth in recent years, and is forecast to show continued expansion. The market is being boosted by increasing demand for natural alternative medicines as a result of disillusionment with some traditional medicines and a growing understanding by consumers of the health benefits of herbals and botanicals. In addition, the increasing use of these 'medicinal' herbs in processed food and drinks is bound to drive the market forward. The success of this market will, however, be influenced by a number of factors. From the supplier side, there are issues of price and calls for an improvement in the standardisation of extracts to consider. The food industry needs to overcome the technological problems sometimes incurred in adding herbals and botanicals to food, which can adversely affect the taste quality of a product. It

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also needs to ensure that the types and levels of 'medicinal' herbals typically being added to food and drinks are both safe and effective, and that any claims made are justifiable Source: International Food Ingredients: 36+, March 2001. ISSN: 0924-5863

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NUTRACEUTICALS INTERNATIONAL 1st January 2001 EUROPEAN MEDICINAL PLANTS MARKET GUIDE. The UK-based Commonwealth Secretariat's Export Market Development Department launched a guide with general information about medicinal plants and their uses at the Medicinal Plants Forum held in Cape Town, South Africa, last year (see also pages 1-3).

The Guide to the European Market for Medicinal Plants and Extracts is one of a series of publications intended to provide market information for the benefit of Commonwealth producers. The purpose is to help exporters of medicinal herbs and plant extracts to better understand the structure and pattern of the European herbal market and to facilitate trade between Europe and the Commonwealth.

According to Denzil Phillips of consultancy firm Denzil Phillips International, who composed the guide, the rapid growth in plant-based medicine is a worldwide phenomenon.

For example, the annual turnover of the top three German herbal manufactures - Schwabe, Madaus and MCH Klosterfrau - is respectively $77.0 million, $69.9 million and $68.7 million. Meanwhile, Hong Kong (with a market worth almost $350 million), the European Union ($250 million) and Japan (with just over $150 million), are the top three worldwide importers of crude medicinal plants, followed by the USA, Germany and Korea. The EU, Hong Kong, Japan and the USA all import more than 50,000 tonnes of crude medicinal plants per annum.

Practical advice

The guide details the growth and development of the European herbal industry, and includes a wealth of practical information for producers and exporters which will help them to gain access to Europe which, with 30% of global sales, is the world's largest market for medicinal plants.

The guide discusses key commercial issues and gives an overview of the structure of the European market, including market size, imports, product prices and consumption, country profiles. Other subjects covered include regulatory issues, protected plants, good agricultural and manufacturing practices and selected product profiles. Various appendices provide information, such as useful books and journals, World Health Organization Medicinal Plant Monographs, European Scientific Co-operative on Phytotherapy monographs and the European herbal monograph.

The Guide to the European Market for Medicinal Plants and Extracts is available for 15 pounds (plus postage and packaging) from Rupert Jones-Parry at the Commonwealth Secretariat, on: fax +44 (0)20 7839 9081; e-mail r.jones-parry@commonwealth.int.

NB – it is also available from Amazon for 20 pounds:

THIS IS THE FULL TEXT: COPYRIGHT 2001 Marketletter Publications Ltd.

COPYRIGHT 2001 Gale Group

©2001 Information Copyright 2000 Gale Group, Inc.

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Copyright 2000 Agra Europe Ltd.

AgraFood East Europe

August 1, 2000 SECTION: Pg. 3 IAC-ACC-NO: 65189050 LENGTH: 258 words HEADLINE: New CEEC market data on medicinal and spice crops. AUTHOR-ABSTRACT: THIS IS THE FULL TEXT: COPYRIGHT 2000 Agra Europe Ltd. Subscription: 270.00 British pounds per year. Published monthly. 80 Calverley Rd., Turnbridge Wells, Kent TN1 2UN., United Kingdom BODY: The German market and price reporting agency ZMP has just produced a new booklet outlining the size and scope of the market for spices, medicinal crops and essential oils in Central and Eastern Europe. The market is a small one, but ZMP notes that it has expanded in size over the past few years as interest in complementary medicine has increased. The CEECs have traditionally been significant producers of these kinds of crops, accounting today more than half of all European exports of such products. The 95-booklet(*) (in German) provides as much as data as is available over production and trade in this sector for a total of 18 Central and Eastern European countries. Major European exporters ZMP notes that more than half of all European exports of medicinal and spice crops originate from the CEECs, with Hungary, Poland, Bulgaria and Albania cited as particularly significant producers. The scale of production is not as large as prior to 1989, when monopolistic agricultural organisations in the CEECs oversaw large-scale production of these crops, but nevertheless a significant export industry still persists. The ZMP publication describes the sector in each of the CEECs, outlining the type of crops grown and the scale of output and trade. A special annex details exports of medicinal and spice products from each of the CEECs in 1997, the latest year for which full data is available.(l) * "Arznei- und Gewurzpflanzen in Osteuropa". Published by ZMP, Rochusstr. 2, 53123 Bonn, Germany. E-mail info@zmp.de. IAC-CREATE-DATE: September 19, 2000 LOAD-DATE: October 04, 2000

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Copyright 2000 Information Access Company, a Thomson Corporation Company;

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Nutraceuticals International

May 1, 2000 SECTION: Pg. NA ; 1362-5411 IAC-ACC-NO: 61971032 LENGTH: 803 words HEADLINE: Europe bans use of Stevia sweetener in foods and supplements. AUTHOR-ABSTRACT: THIS IS THE FULL TEXT: COPYRIGHT 2000 Marketletter Publications Ltd. BODY: The plant Stevia rebaudiana Bertoni, a perennial shrub which is native to certain regions of South America and which has been used for years as a sweetening agent, has now been rejected by the authorities in the European Union for use either as a sweetener or as a novel food ingredient. This herb has recently become popular as a dietary supplement ingredient in the USA, and is widely used as a sweetener in Japan. The saga over the legal status of Stevia in the EU goes back almost five years, when the Scientific Committee for Food of the European Commission rejected an application for Stevia to become a permitted sweetener. The main reason the application was unsuccessful was that the SCF considered that the toxicity data supplied in support of the application were inadequate. New application in1997 When European Regulation No 258/97 on Novel Foods and Novel Ingredients came into force in the EU in 1997, a new application was made for the approval of the plant and dried leaves of Stevia as a novel food ingredient. The application was initially made to the Belgian competent authority, which concluded that the application should be rejected as the applicant had failed to provide adequate data on the safety of the proposed new ingredient. Following the rejection by the Belgian authorities, the application was also considered by the UK Advisory Committee on Novel Foods and Processes. This committee reported at its meeting on September 24, 1998, that it agreed with the opinion of the Belgian authority and also concluded that the applicant had not provided all the information necessary to enable members to make an assessment. The application was also considered by the SCF in Brussels, which published an unfavorable opinion on June 17, 1999. As a consequence of these adverse opinions and the inability of the applicants to provide convincing safety data, the Standing Committee for Foodstuffs of the European Commission took the decision at the end of 1999 to officially endorse the rejection of the applications. This decision has now been formally adopted by the European Commission and published in the Official Journal of the European Communities on March 8, 2000. It says that the Stevia plant or its dried leaves (or preparations thereof) must not be placed on the market within the European Union as a food or food ingredient. Under European law, this also precludes its

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use in dietary supplements sold under food law in any member state of the EU. In the USA, Stevia is sold by a number of companies as a dietary supplement, and also as a sweetener, though it has not been approved under the Food and Drug Administration's Generally Recognized as Safe regulations for food additives. The FDA has turned down three industry requests to use Stevia in foods in the USA, according to David Schardt, an associate nutritionist for consumer watchdog the Center for Science in the Public Interest, writing in the April issue of Nutrition Action Healthletter. The FDA has determined that the data on the substance are not strong enough to support its safety in food. This has led to accusations that the agency was kow-towing to industry, and specifically Monsanto, which markets the hugely-successful artificial sweetener NutraSweet (aspartame) but has now agreed to sell off the business (NI vol 5, no 4). However, despite Stevia's use in Japan, the FDA is concerned that it would be consumed in much higher quantities by Americans if, for example, it was approved for use in diet beverages. Canada has also declined to approve the herb as a food additive. Toxicity concerns Nutrition Action Healthletter notes that while proponents of the herb claim it is side effect-free, laboratory studies have found potential for carcinogenicity and toxicities affecting the reproductive system. For example, when male rats were fed high doses of one of the active components of Stevia, stevioside, for 22 months, sperm production was reduced, the weight of seminal vesicles declined and there was an increase in cell proliferation in the testes, which could result in infertility or other problems. Meanwhile, female hamsters that were fed large amounts of steviol, a compound derived from stevioside, had fewer and smaller offspring. In the laboratory, steviol can be converted into a mutagenic compound, and while this is not known to occur in humans, the phenomenon should be investigated, notes the newsletter. Very large amounts of stevioside can also interfere with the absorption of carbohydrates in animals and disrupt the conversion of food into energy within cells, it adds, noting: "this may be of particular concern for children." The FDA has also received reports of depression, anxiety and hyperactivity in people taking Stevia, notes Reuters. IAC-CREATE-DATE: May 9, 2000 LOAD-DATE: May 10, 2000

EU Phytopharmaceuticals / OTC’s TIAR - Research

October 2002

Information or advice set out in this document is believed to be complete and correct at the time of publication. However, the Australian Trade Commission, its officers, employees, and agents disclaim any responsibility for loss or damage that may be suffered as a result of reliance on this information.

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Herbal Remedies: A World Survey - Market Profiles, Corporate Overview, Company

Profiles and Future Outlook

From EUROMONITOR, Global Market Focus A - July 2001 Table 69 Forecast Value Sales Of Natural Medicines In Germany 2000-2003 DM billion, % constant constant 2000 rsp growth 2000 2.8 - 2001 3.0 5.5 2002 3.2 5.0 2003 3.3 4.5 Source: Euromonitor

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Acknowledgements The author gratefully acknowledges the following support:

1. The Rural Industries Research and Development Corporation for financial support.

2. Mr Tim Groom, Research and Operations Manager, Mr Maurice Kerr, Chief Chemist, and co-workers of Botanical Resources Australia for financial, field and laboratory support.

3. The Tasmanian Institute of Agricultural Research for additional administrative and

financial support and physical resources.

4. Dr Linda Falzari, Mr Craig Palmer, Miss Patricia Saunders and Mr Craig Morris, Tasmanian Institute of Agricultural Research for laboratory and field support.

5. Mr Lyndon Butler and other staff of the Tasmanian Department of Primary Industries,

Water and Environment, for help with field operations at Forthside Research and Demonstration Station.

6. Mr Peter Elliott, Branscombe, Merseylea, and Mr Joe Horak, Waratah, Tasmania for

contributing land for field plots.

Abbreviations BRA Botanical Resources Australia OTCs Over-the-counter (medicines) TIAR Tasmanian Institute of Agricultural Research FRDS Forthside Research and Demonstration Station

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About the author Dr Rowland Laurence is an Associate Professor in the Tasmanian Institute of Agricultural Research, an institute which is a joint venture between the University of Tasmania’s School of Agricultural Science and the Tasmanian Government’s Department of Primary Industries, Water and Environment. Dr Laurence previously managed the Tasmanian Government’s research and extension effort on vegetables and field crops and has worked on the agronomy and nutrition of a range of tropical and temperate horticultural crops. Dr Laurence now leads research projects in the areas of new crops, horticultural agronomy and sustainable farming systems