Production of High Quality Australian Ginseng

Transcription

1 Production of High Quality Australian Ginseng A report for the Rural Industries Research and Development Corporation by R. B. H. Wills & D. L. Stuart December 2001 RIRDC Publication No 01/170 RIRDC Project No: UNC-8A

2 2001 Rural Industries Research and Development Corporation. All rights reserved. ISBN ISSN Production of High Quality Australian Ginseng Publication No. 01/170 Project No: UNC-8A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. 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 Researcher Contact Details Professor Ron Wills Centre for the Advancement of Food Technology & Nutrition The University of Newcastle PO Box 127 OURIMBAH NSW 2308 Phone: (02) Fax: (02) ftrbhw@alinga.newcastle.edu.au RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: rirdc@rirdc.gov.au. Website: Published in December 2001 Printed on environmentally friendly paper by Canprint ii

3 Foreword With the rapidly expanding use of medicinal herbs world-wide, Australia has recognised the opportunity to become an international supplier of many medicinal herbs. As a relatively high cost producer nation the economic benefit will come through the cultivation, processing and marketing of high quality products. High quality in medicinal herbs is the presence of optimal levels of those constituents which confer a health benefit to consumers. In order to support development of a high quality medicinal herb industry in Australia, RIRDC has supported a number of projects under its Essential Oils and Plant Extracts Program. This report details a project on American ginseng that examines changes in the levels of the ginsenosides during plant growth, postharvest handling, processing and in marketed-products. The study identifies a range of options available to maximise the level of active constituents in marketed products. The work detailed in the project was conducted with the active support of the Australian Ginseng Growers Association. This project was funded from RIRDC Core Funds which are provided by the Federal Government. This report, a new addition to RIRDC s diverse range of over 700 research publications, forms part of our Essential Oils 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 purchases at Peter Core Managing Director Rural Industries Research and Development Corporation iii

4 Acknowledgements The authors wish to acknowledge that Ms Xiao-Wei Du performed the laboratory studies which formed part of her PhD program, and Mr Jason van Ritten carried out the grower survey while enrolled in a MSc program, both at the University of Newcastle. We would also like to thank the Australian Ginseng Growers Association (AGGA), and in particular Charlene and Fred Hosemans, and the Committee of AGGA for their generous and active support of research into the quality of American ginseng at the University of Newcastle. Thanks are also given to Dr David Evans, Program Manager, RIRDC for valued suggestions and encouragement during the course of the project and to Dr Krystyna Johnson for her involvement in the grower survey. iv

5 Contents Foreword iii Acknowledgements... iv Executive summary... vi 1. Introduction Objectives Analysis of ginsenosides Neutral ginsenosides Malonyl ginsenosides Changes in neutral ginsenosides during plant growth Variation between individual roots Changes in plant parts over a growing season Neutral ginsenosides composition Concentration of neutral ginsenosides Total amount of neutral ginsenosides Changes in neutral ginsenosides with root age Variation in ginsenosides in roots grown at different sites Processing and storage Drying temperature Blanching Alcoholic extract Spray Drying Neutral ginsenosides in commercial products Farmer survey of growing Practises Farm environment Growing practices Grower circumstances Conclusions and recommendations Need for analysis of active constituents Benefit of maximising root size Need for improved postharvest operations Quality of retail products Development of the Australian industry References v

6 Executive summary American ginseng (Panax quinquefolium) is a medicinal herb native to the temperate regions of North America. It has found considerable usage in north Asia where Asian ginseng (Panax ginseng) has been a traditional medicine for many centuries. The accelerating affluence of many Asian countries coupled with the growing popularity of alternative therapies in the Western world has led to a marked increase in the international trade in both species of ginseng. The growth in ginseng trading has led to the establishment of an Australian industry based on the growing of American ginseng. While the industry is still in its infancy, it has created an umbrella organisation, the Australian Ginseng Growers Association (AGGA), which is promoting a national approach to industry development. Australia is agriculturally well positioned to capture a share of the world market and cropping is now conducted on organic principles in a wide range of regions across the eastern and southern States. If Australia is to become successful at exporting and import substitution, it needs to resolve various marketing and quality issues. As traders and consumers become more sophisticated in their requirements for product quality, and the world crop supply increases to match, or as appears likely to exceed market demand, there will be greater competition in the ginseng market. Countries which have the reputation and ability to consistently supply high quality raw material and processed products will gain preferential access to the higher price market segment, thus maximising the economic return from the crop. The ultimate determinant quality factor in all medicinal herbs, including ginseng, is the concentration of active constituents that impart a health benefit to the human body. While a number of groups of active compounds have been identified, it is widely accepted that the saponins known as ginsenosides, are major active constituents in ginseng. The research studies described in this report used the major ginsenosides of Rg1, Re, Rb1, Rc, Rb2, and Rd as the markers of ginseng quality. The overall aim of the project was to assist the Australian growers to develop a marketing strategy for future Australian-grown American ginseng crops based on quality. The research objectives focused on determining: reliable methods for the analysis of ginsenosides, changes in ginsenosides in plant parts during plant growth and maturation, effect of postharvest handling and processing operations on ginsenosides, quality of ginseng products available in retail outlets, and survey the situation and practices of Australian ginseng farms. Efficient and reliable quantitative analytical methods for the analysis of both neutral and malonyl ginsenosides in American ginseng were developed using high performance liquid chromatography (HPLC). The importance of neutral ginsenosides is well known but relatively recently recognition of the importance of malonyl ginsenosides due to their potential hydrolysis to neutral ginsenosides has highlighted the need for both malonyl and neutral ginsenosides to be analysed in any quality assurance system established by the industry. In this project, the importance of malonyl ginsenosides was not recognised until after most of the growth trials had been completed and hence data for malonyl ginsenosides was mostly only obtained for the processing and storage trials. The neutral ginsenosides in the various parts of 4-year old American ginseng plants grown in Victoria were determined at seven harvest periods over a whole growing cycle from leaf emergence to dormancy. It was found that ginseng roots contained a similar composition and concentration of neutral ginsenosides as roots grown in North America and Asia and should therefore be as acceptable in medicinal quality for the international market as American ginseng grown in other countries. In addition, the concentration of ginsenosides tended to be at the upper level of the range previously reported for American ginseng. The cultivation of ginseng under a canopy of eucalypt trees has thus appeared not to have a detrimental effect on the accumulation of neutral ginsenosides. vi

7 The concentration of the neutral ginsenosides over the growth period showed little change in the main root and lateral root. The highest concentration of ginsenosides was in the leaf and the hair root and in these plant parts, the concentration significantly increased to a maximum value at the growth stage when green fruit was present. The absolute amount of neutral ginsenosides in plant sections, the amount being a function of plant weight and ginsenosides concentration, showed that the main root and lateral root contained most of the plant ginsenosides due to their high contribution to total plant weight. The hair roots had the highest ginsenosides concentration but contributed only about 5% of the total ginsenosides due to its relatively low weight. However, the leaves when fully developed contained about 25% of the total ginsenosides from a high concentration and weight contribution. The ginseng industry world-wide has traditionally marketed only the main and larger lateral root sections. The findings from this project suggest that utilisation of the leaves and, to a lesser extent, the hair roots, could generate a valuable by-product with beneficial medicinal properties. Commercial utilisation of leaf material would, however, require harvesting of the plant before the leaves started to senesce rather than at the current dormant stage. This would appear feasible since there was no reduction in the concentration of ginsenosides in roots if harvested at an earlier stage of plant development. The pharmacological activity of the two plant sections would appear to be similar as the composition of ginsenosides is not radically different between the leaf and root but this would need further investigation. An additional consideration would be to confirm that early harvesting of the leaf every year did not affect growth of the root. The harvesting of leaves would at least allow some return to growers before the root attained commercial maturity. The determination of ginsenosides in roots of different age grown on a single site and of the same age but grown in different locations showed that for both sets of roots, there was a strong linear relationship between root weight and ginsenosides concentration. The findings suggested that it would be advantageous for the industry to identify growing practices or improved plant nutrition that increase the rate of root growth. The benefits to be gained would not only be an increase in root size but also an increase in ginsenosides concentration. While there was only a limited number of samples in the survey of roots from different farms, the results also suggested that field cultivated material under artificial shade produce a faster growing root and therefore a greater concentration of ginsenosides than forest-grown roots. This relationship needs to be explored further. In the farm survey, the malonyl ginsenosides were also analysed and the data show that in fresh roots the proportion of neutral to malonyl ginsenosides was in the ratio of 3:2. This factor could be useful in estimating the total ginsenosides in roots where the malonyl ginsenosides were not analysed. Since the importance of managing postharvest handling operations in maintaining ginseng quality does not appear to have been extensively studied in other countries, there is an opportunity for Australia to gain a market advantage by retaining ginsenosides in traded products through improving such practices. The project examined the effect on ginsenosides of the drying of fresh root, storage of dried root, steam blanching of fresh root, alcoholic extraction of dried root powder and spray drying of the alcoholic extract. A study was conducted to determine the effect of air temperature on the ginsenosides during the drying of fresh roots in a hot air drier. Increasing the drying temperature was found to cause a loss of malonyl ginsenosides, a small increase in neutral ginsenosides and a loss of total ginsenosides. The drying temperature should therefore be minimised to optimise retention of total ginsenosides. A drying temperature of 55ºC is recommended over lower temperatures due to the reduced drying time with little loss of ginsenosides. Drying at higher temperatures further reduces drying time but with enhanced loss of ginsenosides along with significant tissue browning. While there was no evaluation of other drying technologies such as heat pump, low pressure and freeze drying, it is suggested that these technologies would result in reduced loss of ginsenosides as they employ less heat during drying, but the equipments are much more expensive to purchase and operate. vii

8 Storage of dried ginseng root powder over the temperature range of 5 to 30 C was found to be temperature dependent. There was increasing loss of total ginsenosides with increasing temperature which was associated with an initial conversion of malonyl ginsenosides to the corresponding neutral ginsenosides. Optimum storage for dried material is therefore at low temperatures such as 5 C where no appreciable loss of ginsenosides occurred over three months. However, it would also seem prudent based on experience with other medicinal herbs, to use packaging that protects the product from absorption of atmospheric moisture and probably also light. Steam blanching is a traditional method to produce the "red ginseng from Asian ginseng root. No published studies are available to show the change in malonyl and neutral ginsenosides during the blanching of Asian or American ginseng but this study found a similar loss of ginsenosides with increased blanching time as with increased heat loading during drying. At the optimum blanching time of 2 hr which produces an attractive appearance for the red ginseng product, there was a loss of 15% in total ginsenosides which is considered acceptable. From a quality perspective, the production of red American ginseng is worthy of further investigation by the Australian industry. The establishment of an Australian processing industry for ginseng requires an efficient method of extraction of ginsenosides and their subsequent concentration into a saleable product. Ethanol is a common, acceptable solvent used for the production of a wide range of extracts in the food and pharmaceutical industries. This project found that a relatively wide range of ethanol/water mixtures was able to extract about 85-90% of both neutral and malonyl ginsenosides. The use of 50% ethanol is considered to be the optimal concentration. The project examined spray drying as a representative method of concentrating the alcoholic extract. It was found that using commercially acceptable spray drying temperature regimes, the resulting dried product had a highly acceptable colour and texture with less than a 15%. loss of ginsenosides. It is considered that a market exists for the sale of dried ginseng extract for incorporation into other food or pharmaceutical products. To examine the quality of ginseng products available to consumers, a range of dried and processed ginseng products were purchased from retail outlets and the concentration of neutral ginsenosides determined. Considerable variation was found to exist between products. This variation would arise from different amounts of added ginseng into a product and natural or induced variability between batches of raw material during postharvest operations. Unlike previous similar analysis on other medicinal herbs, there were no products with near zero levels of ginsenosides. However, each of the product classes contained varying concentrations of ginsenosides. The highest concentration was found in root powders and tea bags followed by the dry root and tablets/capsules. The higher values in root powders and tea bags probably reflect the use of hair and smaller lateral root sections which have a higher ginsenosides concentration. The wide range of values found for the tablet and capsules would reflect varying efficiencies of processing as well as variable quality of raw material. The variation within each product class does illustrate the need for growers and processors to exercise quality management of postharvest handling and processing operations in order to standardise on raw material inputs and minimise post-farm gate losses of ginsenosides. From a consumer perspective, the findings highlight the need for improved labelling of products. While many products do contain high levels of ginsenosides they cannot be identified by purchasers. Product labels should contain a more standardised format on ginseng content and include the concentration of nominated active constituents in the final product, similar to processed foods. This information should be supplied both on a unit product weight or volume, as well as on a recommended dose basis. While this project was on quality management issues, interaction with the Australian and international ginseng industry has generated some ideas on how the Australian industry might develop. Since Australia can only be a small contributor to the international ginseng market, it would benefit from having a national ginseng brand and a national marketing and processing operation. This would allow the many small growers to equal access international markets with branded and guaranteed high quality products that could attract a price premium. Such an operation would further assist the economic viability of the industry by allowing the production of commercial products from non-root plant sections such as hair root and leaf. The existence of AGGA offers a ready platform that could viii

9 develop beyond a grower consultative body to become the national marketing and processing organisation. ix

10 1. Introduction The World Health Organisation has claimed that 80% of the world population uses medicinal herbs for beneficial health effects. While medicinal herbs are intrinsically enmeshed in many cultural practices, the use of medicinal herbs over recent decades in Western countries has been growing at an extraordinary rate. This is despite the development of pharmaceuticals achieving substantial advances in alleviating suffering and prolonging life. The interest in medicinal herbs in the West is primarily due to the disillusionment by consumers with medical practices, and indeed with modern technology in general. Increasing use in Asian countries has also occurred due to greater affluence allowing access to previously expensive remedies. Medicinal herbs were traditionally obtained by harvesting plants from natural woodlands and fields. The slow development of a cultivation industry with the continuing reliance on a diminishing source of wild herbs has seen the demand for many herbs greatly exceed supply with resultant substantial price rises. In addition, the market for medicinal herbs in the West is based around processed products resembling those from the pharmaceutical industry which demands a high level of quality control. This has created the need for research to determine the effects of cultivation and processing on the quality of medicinal herbs. Ginseng consists of two major varieties, Panax ginseng (Asian) and Panax quinquefolium (American), with the common name based on their respective native geographical location. American ginseng is a perennial, herbaceous plant of the Araliaceae family, and along with Asian ginseng, traditional use is based on root material. American ginseng has been commercially farmed for the last 100 years in North America and within Asia. Apart from some differences in the processing of roots, both varieties are now used although for different medicinal purposes. Ginseng roots grow in cold climates under heavy shade and is therefore characterised by long growing periods and very low weight returns per annum. Traditional quality characteristics for the Asian ginseng industry can differ throughout Asia, however, common themes involve root shape and colour, with the emphasis on the main and lateral root sections. Processing of ginseng utilizes the main root and discards much of the small lateral and hair roots, and all the aerial sections. Consequently, much research has been completed to determine the optimum farming techniques that will maximise the shape and size of the root. Hong Kong is the traditional distributor for the international trade in ginseng. The growth in the ginseng industry is seen by the Hong Kong market increasing from 3895 t in 1990 to 5132 t in 1997 (Sadler, 1999). Imports into Hong Kong and Taiwan now have a value of about US $350 million. China produces half of the world ginseng with South Korea contributing 32% and the United States 8%. Ginseng imports to Australia have shown a similar increase from 4 t in 1989 to 17 t in The price of ginseng root varies according to a range of factors and can command prices of about A$6000/kg for excellent upper bubble root to about A$400/kg for #3 red trunk root (Sadler, 1999). In Australia, high quality 7-year-old roots have sold for about $1000/kg (Hosemans & Hosemans, 1996). In response to the increased worldwide demand, American ginseng has been successfully cultivated in Australia for the past 10 years. Production of ginseng in Australia is difficult to ascertain but it is estimated that about 150 ha is currently under cultivation with most of the crop destined for export. Australia is agriculturally well positioned to capture a share of the 1

11 international market and cropping is now conducted in a wide range of regions across the southern States. If Australia is to become a successful long-term exporter and to replace imports with locally grown material, it needs to resolve various marketing and quality issues. It is difficult for high cost production countries such as Australia to compete with low cost developing countries on price. However, the Australian wheat industry has shown that it is possible to be internationally competitive on quality. It would seem that the Australian ginseng industry should aim to develop an international reputation as a supplier of high quality raw material as well as processed and manufactured products in order to gain preferential access to the higher price market segment. This will assist in obtaining continuing sales and an adequate economic return from the crop. A major factor in the determination of quality in medicinal herbs is the concentration of those constituents that lead to a health benefit. A considerable number of compounds in ginseng have been identified as being active constituents, however, a group of saponins now known as ginsenosides has been shown in immunological testing to be the most important quality markers. (Li et al., 1996). Until recently, the neutral ginsenosides were the primary group investigated, however, the discovery of the malonyl ginsenosides and development of appropriate quantitative analytical methods has indicated the need to determine the levels of both groups. This is due to the malonyl ginsenosides converting to the neutral ginsenosides as the first step of human metabolism, thereby increasing the effective ginsenosides concentration (Awang, The research studies described in this report used total ginsenosides as the marker for determining quality. The early growth studies examined only the neutral ginsenosides but later growth studies and all the processing studies also determined the malonyl ginsenosides. 1.2 Objectives The overall aim of the program was to develop quality parameters and associated tests to enable growers to harvest and handle American ginseng to maintain optimum quality, and to identify efficient processing techniques that ensure optimum quality is transferred through to the end products. This was pursued experimentally by determining: reliable methods for the analysis of neutral and malonyl ginsenosides, optimum plant sections and harvest times to maximise levels of ginsenosides, quality of ginseng roots currently traded from Australian farms, effect of postharvest handling practices of fresh material on the levels of ginsenosides, effect of processing operations involved in the manufacture of value-added products on levels of ginsenosides, and levels of ginsenosides in retail products available to consumers. The research program was conducted in close liaison between The University of Newcastle and the Australian Ginseng Growers Association. Apart from educating the researchers in ginseng industry practices, this liaison ensured the individual projects retained industry relevance and assisted in the transfer of findings to industry. 2

12 2. Analysis of ginsenosides 2.1 Neutral ginsenosides Analysis of neutral ginsenosides was achieved by HPLC using the method of Ma et al. (1996). Separation was achieved with a reversed phase column, a mobile phase gradient of water (A) and acetonitrile (B) with 0-20 min being 20-22% B, min from 22-46% B at 1.5 ml/min, column temperature of 40 C and peak detection by UV at 203 nm. Figure 1 is a chromatogram of the six neutral ginsenoside standards (Rg1, Re, Rb1, Rc, Rb2 and Rd) which were used to determine the separation characteristics. Figure 2 is a chromatogram showing a separation of ginsenosides in a root extract and the presence of the six neutral ginsenosides is easily discernable and exhibit a good correlation with the separation achieved by Ma et al. (1996). Figure 1 HPLC chromatogram of neutral ginsenosides (1: Rg1, 2: Re, 3: Rb1, 4: Rc, 5: Rb2, 6: Rd) min 3

13 mabs min Figure 2 HPLC chromatogram of neutral and malonyl ginsenosides in American ginseng root extract. (1: Rg1, 2: Re, 3: Rb1, 4: Rc, 5: Rb2, 6: Rd, 7: mrb1, 8: mrc, 9: mrb2, 10: mrd) The method for extraction of ginsenosides from dried ginseng material was to grind the plant part to a powder and actively mix with an organic solvent. A range of particle sizes, methods of mixing, and solvents were extensively evaluated to determine the optimum method for extraction. On the basis of these studies, the method adopted was to grind a dried sample to pass through sieve mesh No.60 (<250 µm), extract with 80% methanol by sonicating for 15 min at room temperature, followed by filtration through filter paper. The extraction was repeated two additional times and the combined extracts were made up to volume. The efficiency of ginsenosides recovery was determined by adding a ginsenoside standard (Rb1) solution into a root sample and extracting, along with a control root sample; >99% of the added ginsenoside was recovered in the analysis. The method was highly reproducible with a coefficient of variation between analyses of about 2%. 4

14 The extraction of ginsenosides from fresh material needed to take into account the presence of a high water content. Following a similar extensive evaluation of the extraction method as for dried material, the method adopted to extract ginsenosides from fresh material was to homogenize a fresh ginseng sample with 100% methanol at high speed for 3 min, sonicate for 15 min followed by filtration. The extraction was repeated two additional times and the combined extracts made up to volume. 2.2 Malonyl ginsenosides The malonyl ginsenosides were determined with an indirect method due to standards not being available. HPLC analysis was performed twice for each sample. The first analysis quantified the six neutral ginsenosides in the extract solution. The extract was then hydrolyzed to convert the malonyl ginsenosides to their respective neutral ginsenosides. A second analysis quantified the neutral ginsenosides in the hydrolyzed extract. Identification of the malonyl-ginsenoside mrb1, mrc, mrb2, mrd was carried out by comparing the chromatogram of hydrolyzed extract with that of the original extract, and also with that reported by Court et al. (1996). The concentration of individual malonyl ginsenosides in the original extract was calculated by subtracting its relevant neutral ginsenoside concentration in the original extract from that in the hydrolyzed solution. The HPLC system adopted was that of Court et al. (1996) which was similar to that used above for the neutral ginsenosides except that a phosphate buffer was the aqueous phase. Figure 2 shows the separation achieved for the four major malonyl ginsenosides, mrb1, mrc, mrb2, and mrd, in a root extract and their elution characteristics in relation to the six neutral ginsenosides. Hydrolysis was performed by removing the methanol in original extract then mixing the residue with 5% potassium hydroxide solution. The hydrolyzed solution was neutralized with potassium hydrogen phosphate and made up to volume with acetonitrile. 5

15 3. Changes in neutral ginsenosides during plant growth Studies were conducted to determine changes in the neutral ginsenosides between: individual roots, different plant parts over a growing season roots of different age, and roots from different growing sites. 3.1 Variation between individual roots The variation in neutral ginsenosides concentration between single roots was analyzed on dry and fresh roots. The data in Table 1 show that the variation in the concentration of neutral ginsenosides between 10 individual 4-year-old ginseng roots that were dried at 40ºC was large with values for total ginsenosides ranging from mg/g dried root, with a mean value and standard of deviation of 56±17. There was a similar variation for individual ginsenosides. Fourteen 4-year-old fresh roots were individually analysed. The data in Table 2 show that the variation in ginsenosides concentration between fresh roots was large and similar to the above data with values for total ginsenosides ranging from mg/g dried root, with a mean and standard deviation of 54±12. A similar variation existed for individual ginsenosides. Table 1 Neutral ginsenosides concentration in individual dried roots Neutral ginsenosides concentration (mg/g dried root) Root Rg 1 Re Rb 1 Rc Rb 2 Rd Total Mean SD ±1.3 ±5.3 ±11.0 ±0.9 ±0.1 ±1.0 ±17.0 Table 2 Neutral ginsenosides concentration in individual fresh roots 6

16 Neutral ginsenosides concentration (mg/g dried root) Root Rg 1 Re Rb 1 Rc Rb 2 Rd Total Mean SD ±4.7 ±5.0 ±8.5 ±0.8 ±0.1 ±1.0 ± Changes in plant parts over a growing season Four-year old ginseng plants were harvested from 2 growers in the Dandenong district, Victoria. The plots were selected at each location before plant germination. Seven harvest times were selected based on the visible plant growth stage. These stages and the harvest dates were: Stage 1: sprouted leaf, Stage 2: distinctive separation of leaf and stem, Stage 3: plant in full flower, Stage 4: green fruit set, Stage 5: red fruit present, Stage 6: fruit has abscised and leaf senescence has commenced, Stage 7: aerial parts have totally senesced. At each harvest, two replicate samples of 5 individual plants were randomly obtained from each grower throughout the plots. Each group of plants was separated into 7 sections comprising main root, lateral root, hair root, leaf, stem, flower and fruit. Each section was analyzed for neutral ginsenosides Neutral ginsenosides composition 7

17 Tables 3 and 4 give the proportion of individual ginsenosides present in root and aerial plant sections, respectively. These were obtained over the growing season and averaged over both growers. The data show that Re was the major ginsenoside in both root and aerial sections, while Rb1 was also a major component in the root and Rd in the aerial section. In the root sections, Rb1 and Re were, on average, each present at about 40% of total ginsenosides with Rg1, Rc, Rb2 and Rd each present at <10% although the composition of the hair root differed slightly to that of the main and lateral roots. The aerial sections comprised about 50% Re, 30% Rd and 10% or less of the other ginsenosides. Rd was the only ginsenoside in the root to show a significant change over the season. Figure 3 shows that the composition of Rd followed a quadratic relationship in all root sections over a growing season with the maximum composition occurring when green and mature fruit were present on the plant. In the total aerial plant parts, the proportions of all neutral ginsenosides except Rc changed significantly over the season. Re decreased from about 60% at germination to about 30% at flowering, then increased to about 40% at the aerial senescence stage. However, the proportion of Rd increased from about 10% at germination to about 40% at flowering, then fell to about 25% at the aerial senescence stage. The same changes for both compounds were reflected in leaf and stem. Figure 4 shows the changes in Rd in aerial sections over the growing season. These results are consistent with the findings of Li et al. (1996) who tested leaf and whole root material from American ginseng grown in nine locations across British Columbia, Canada and those of Smith et al. (1996) for main and hair root. 8

18 Table 3 Neutral ginsenosides composition in root sections over a growing season % of total neutral ginsenosides Plant section Harvest stage Rg 1 Re Rb 1 Rc Rb 2 Rd Whole root Mean LSD (5%) NS NS NS NS NS ± Main root Mean LSD (5%) NS NS NS NS NS ± Lateral root Mean LSD (5%) NS NS NS ±1.6 ±0.2 ± Hair root Mean LSD (5%) NS ±3.8 NS NS NS ±3.8 Harvest stage 1: leaf sprout; Stage 2: separation of leaf and stem; Stage 3: full flower; Stage 4: green fruit; Stage 5: red fruit; Stage 6: some leaf senescence; Stage 7: full aerial senescence Table 4 Neutral ginsenosides composition in aerial plant sections over a growing season 9

19 % of total neutral ginsenosides Plant section Harvest stage Rg 1 Re Rb 1 Rc Rb 2 Rd Total aerial Mean LSD (5%) ±3.1 ±7.2 ±3.1 NS ±5.3 ± Leaf Mean LSD (5%) ±3.4 ±6.8 ±3.0 ±2.2 ±5.8 ± Stem Mean LSD (5%) ±4.0 NS ±4.6 NS NS ±4.3 Flower Fruit Mean LSD (5%) NS ±5.2 NS ±3.0 ±2.8 ±1.5 10

20 Whole root 20 y = x x (R 2 = 0.60) Main root 20 % Rd 10 0 y = x x Lateral root 20 y = x x Hair root 20 y = x x Growth time (days) Figure 3 Change in composition of Rd in root sections over a growing season 11

21 Total aerial 45 y = x x Leaf 50 y = x x % Rd Stem y = x x Growth time (days) Figure 4 Change in composition of Rd in aerial sections over a growing season 12

22 3.3.2 Concentration of neutral ginsenosides The concentration of neutral ginsenosides (Table 5) behaved differently between plant sections over the seasonal growth period. The data show the concentration in the leaf significantly increased while that in the flower/fruit decreased. The main root, lateral root and stem did not show any significant change in concentration while the hair root showed a significant maximum at stage 4 (green fruit) before becoming slightly lower. The combined root sections showed no significant increase, however, the combined aerial sections and the combined plant sections significantly increased over the growing season. The significant increases which occurred in the hair root were due to increases in Re and Rb1 (33% each), and Rd and Re increasing by 15%. The increase in leaf concentration was primarily influenced by Rd (46%), and increases in Rb2 and Re (each about 20%). The significant decreases in the reproductive system of the flower and fruit were due to Re (45%), Rd (19%) and Rb2 (16%). The level of total neutral ginsenosides in the main roots of mg/g is well within the range reported for American ginseng roots grown in North America of mg/g (Konsler et al. 1990; Li et al. 1996; Court et al. 1996; Smith et al. 1996; Reynolds 1998; Wang et al. 1999). However, the ginsenosides concentration reported by Li et al. (1996) and Konsler et al. (1990) for leaf of mg/g is lower than the current findings of about 60 mg/g for mature leaf. This is the first reported data for ginsenosides in flower, fruit, and stem sections of American ginseng. The root hair has been known to contain a higher concentration of ginsenosides (Liberti and Der Marderosian 1978; Smith et al. 1996) which has been proposed due to the higher concentration found in the periderm and cortex root tissues (Tani et al. 1981). Table 5 Concentration of total neutral ginsenosides in plant sections during growth Stage Main root Lateral root Hair root Total ginsenosides concentration (mg/g) Leaf Stem Flower/ Fruit Total Aerial Total Root Total Root + Aerial * * * * LSD *- Significant increase (p<0.05) - Significant decrease (p<0.05) 13

23 3.2.3 Total amount of neutral ginsenosides The accumulation of ginsenosides is a function of the plant section weight and its ginsenosides concentration. The data in Table 6 show a significant increase in the amount of ginsenosides in the main and lateral roots and leaf during the season. While the ginseng industry has focused on using the root sections, Table 6 indicates that the total aerial content constitutes approximately 30% of available ginseng when green fruit were present (stage 4), and this is dominated by the leaf content (85% of total aerial content). Hair root contains between 5-15% of ginsenosides content. Since the concentration of ginsenosides in root sections did not markedly change with time, the total amount of neutral ginsenosides was therefore mainly affected by an increase in dry matter content. The concentration in whole root was significantly higher than in the total aerial part while in root sections, the concentration was hair root > lateral root > main root, and in aerial parts was leaf > flower & fruit > stem. Due to its large weight, the main root contained about 40% of the neutral ginsenosides in whole plant with the lateral root containing 34% and the leaf 19%. Table 6 Neutral ginsenosides content in each plant section during growth Stage Main root Lateral root Total ginsenosides content (mg/plant section) Hair root Leaf Stem Flower/ Fruit Total Aerial Total Root Total Root + Aerial * 30.8* * * 115.5* 171.3* * * 70.3* 169.9* 240.2* * LSD *- Significant increase (p<0.05) - Significant decrease (p<0.05) 3.3 Changes in neutral ginsenosides with root age The increasing age of ginseng roots has traditionally been used as a measure of quality, and the literature shows trends of a linear increase in weight and concentration over 6 years of growth indicating potential benefits of long-term cultivation for greater yield of total ginsenosides. This study was conducted on plants up to 13 years in age obtained from the only farm in Australia with roots of this age range in order to determine the commercial viability of long term cultivation and the results are given in Table 7. Figure 5 shows that a quadratic relationship existed between ginsenosides concentration and root age with a maximum concentration in 8 year old roots. It 14

24 also shows a linear relationship between root weight and root concentration. This indicates that concentration is primarily a factor of weight rather than root age. This suggestion that ginsenosides concentration is a primarily a function of root weight rather than root age has been a speculation raised in previous publications (Soldati and Tanaka 1986; Court et al. 1996). However, this is the first study to show roots with higher concentrations at a younger age. Previous studies on root age have not extended beyond 6 year old roots, but it is well known that wild crafted ginseng roots increase in size over many years. The older roots sampled in this study were the few remaining plants from limited early experimental plantings in Australia and are probably not representative of a normal well cultivated crop. It is assumed that the smaller root size of the older roots was due to these being the remaining few plants left in the plot with the larger roots having been harvested in earlier years. The results do, however, emphasize the strong relationship between root size and ginsenosides concentration. Table 7 Influence of root age on neutral ginsenosides and root weight. Root Age Weight (g) Ginsenosides conc. (mg/g) Ginsenosides content Ginsenosides production/year

25 60 50 Concentration (mg/g) y = 9.2x x Root age (years) Concentration (mg/g) y = 4.33x Root Weight (g) Figure 5 Relationship between neutral ginsenosides concentration and root age and root weight 16

26 3.4 Variation in ginsenosides in roots grown at different sites The variation of total neutral and malonyl ginsenosides concentration in ginseng root of the same age but grown at different sites was examined on fresh ginseng roots (3 year old) obtained from 10 ginseng growers in Australia and New Zealand. Three replicate samples of 3 individual roots from each ginseng grower were cleaned, dried, ground to <250 µm and analysed for ginsenosides. The analytical data were divided to three groups according to whether the roots were grown under forest with natural tree shade, forest with natural tree and artificial shade or a field with artificial shade. The data in Table 8 show that there was a significant difference in the concentration of ginsenosides due to the growing conditions. The root weight, concentration of neutral, malonyl and total ginsenosides and total amount of ginsenosides of roots grown in a field with artificial shade were higher than in roots grown in a forest with natural or artificial shade. The interrelationship of root weight with ginsenosides is depicted in Figure 6 and shows a linear correlation between root weight and concentration of ginsenosides. The results suggest that techniques which increase the rate of root growth will also increase the concentration of ginsenosides. Table 8 Root weight and ginsenosides level of roots grown in different conditions n Dry weight Ginsenosides Concentration (mg/g dried root) Total amount Growing condition (g per root) Neutral Malonyl Total (mg per root) Forest Forest & artificial shade Field & artificial shade LSD (5%) ±1.9 ±6.1 ±7.2 ±11.9 ±119.5 Ginsenosides concentration (mg/g) 100 y = 3.83x Total 60 y = 1.71x y = 2.13x Root weight Neutral Malonyl Figure 6 Correlation of root weight with ginsenosides concentration in roots of the same age grown at different sites 17

27 4. Processing and storage Changes in neutral and malonyl ginsenosides of ginseng root were determined during drying and blanching of fresh root, storage of dried root powder and obtaining and then spray drying of an ethanolic root extract. The levels of neutral ginsenosides only was determined in commercial retail products, as this was an early study. 4.1 Drying temperature The effect of drying temperature on moisture loss and ginsenosides concentration in ginseng root was examined. on 4 year-old roots from a farm in Victoria. The roots were washed then dried at 40º, 55º and 70ºC using a hot air dryer. There were a small number of roots in the dryer so there was no restriction on air movement around the roots. Drying was terminated when the water content in samples was <10% which is a commercially dry root. The time taken to dry roots in the hot air dryer was found to be temperature dependant and the rate of drying at the various temperatures and the time to dry to 90% water loss are shown in Figure 7. The time taken to dry to 90% moisture loss was five times longer at 40ºC than at 70ºC and three times longer at 40ºC than at 55ºC. After drying at 70 C, the root colour was observed to be darker than for roots dried at lower temperatures but there was no discernible difference between the colour of roots dried at 55º and 40ºC. The data in Figure 7 show that the concentrations of total ginsenosides and malonyl ginsenosides in dried root decreased with an increase in drying temperature but the concentration of neutral ginsenosides increased. This suggests that heat induces some hydrolysis of malonyl ginsenosides to neutral ginsenosides which is also degrading as reflected in the loss of total ginsenosides. The loss of total ginsenosides was about 18% as the drying temperature increased from 40ºC to 70ºC. 4.2 Blanching Steam blanching of fresh Asian ginseng root (white ginseng) has been used for many centuries in China to produce red ginseng which is firstly more resistant to degradation during storage but also has different pharmacological properties and hence different medicinal uses (Hideaki, 1999). The effect of steam blanching on the ginsenosides in fresh root was examined with 3-year old roots harvested in Victoria. Blanching was undertaken using a traditional method where roots were placed in a bamboo basket and steamed for 1-4 hr. The steamed samples were dried at 40ºC. Regression analysis (Figure 8) showed that the concentration of neutral ginsenosides initially increased during blanching up to 2 hr but decreased slightly on longer blanching time while the level of malonyl ginsenosides decreased with increasing blanching time and attained near zero levels after 3 hr blanching. The concentration of total ginsenosides linearly decreased with increasing blanching time. No ginsenosides were detected in the blanch water hence the loss was not due to leaching. The roots blanched for 2-4 hr had an appearance similar to red Asian ginseng. 18