High Quality Australian Skullcap Products

Home , Scutellaria, Scutellaria galericulata, Scutellaria lateriflora

Generation of High Quality Australian Skullcap Products

A report for the Rural Industries Research and Development Corporation

by R.B.H. Wills and D.L. Stuart

March 2004

RIRDC Publication No 04/020 RIRDC Project No UNC-6A

ISBN 0642 58730 2 ISSN 1440-6845

Generation of High Quality Australian Skullcap Products Publication No 04/020 Project no. UNC-6A

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 Communications Manager on phone 02 6272 3186.

Researcher Contact Details Professor R.B.H. Wills School of Applied Sciences, University of Newcastle, Ourimbah NSW 2258

Phone: 02 43484140 Fax: 02 43494565 Email: [email protected]

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 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604

Phone: 02 6272 4819 Fax: 02 6272 5877 Email: [email protected] Website: http://www.rirdc.gov.au

Published in March 2004 Printed on environmentally friendly paper by Canprint

FOREWORD

The use of medicinal herbs is expanding worldwide and Australia is actively seeking to capitalise on the opportunity to become an international supplier of many medicinal herbs. Since Australia is a relatively high cost producer nation, economic benefit will only be derived through the growing and marketing of high quality products. High quality in medicinal herbs ultimately relates to the presence at high levels of those constituents that 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 skullcap (Scutellaria lateriflora) that examines the levels of key active constituents of skullcap in different genetic stock, during plant growth, postharvest handling and processing and in marketed-products. The study identifies a range of options available to maximise the level of active constituents during the production and marketing chain.

The work detailed in the project was commenced with the active support of the skullcap grower and processor, Subiaco Herbs Pty. Ltd, Walcha NSW.

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

This report, an addition to RIRDC’s diverse range of over 1000 research publications, forms part of our Essential Oils and Plant Extracts R&D program, which aims to support the growth of s profitable and sustainable essential oils and natural 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/fullreports/index.html • purchases at www.rirdc.gov.au/eshop

Dr Simon Hearn Managing Director Rural Industries Research and Development Corporation

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ACKNOWLEDGMENTS

The authors wish to thank Philip and Katie Brown at Subiaco Herbs for their active support in the initiation of research and their technical, financial and materials contribution to the conduct of the skullcap project. Thanks are also given to Ms Shona English and Ms Jeanine De Diana for conducting laboratory and field studies.

Contents

FOREWORD...... iii ACKNOWLEDGMENTS...... iv EXECUTIVE SUMMARY...... vi

1. INTRODUCTION...... 1

2. ANALYSIS OF FLAVONOIDS...... 3

3. FLAVONOIDS IN SKULLCAP DURING GROWTH ...... 8 3.1 Flavonoids composition ...... 8 3.2 Level of flavonoids in plant sections...... 9

4. POSTHARVEST HANDLING AND DRYING ...... 10 4.1 Physical damage to freshly harvested plants...... 10 4.2 Delay between harvest and drying ...... 11 4.3 Drying temperature...... 11 4.4 Storage of dried material ...... 12

5. EXTRACTION AND PRODUCT QUALITY...... 13 5.1 Effect of ethanol:water ratios ...... 13 5.2 Analysis of commercial products ...... 14

6. DISCUSSION AND RECOMMENDATIONS ...... 16 6.1 Need for analysis of flavonoids...... 16 6.2 Variation in flavonoids between plant sections...... 16 6.3 Need for improved postharvest handling practices ...... 16 6.4 Need for improved processing operations...... 17

7. BIBLIOGRAPHY ...... 18

EXECUTIVE SUMMARY

Skullcap (Scutellaria lateriflora) is a medicinal herb plant native to North America and its aerial parts are used as a sedative in form of herbal teas, tablets, capsules and oral liquid preparations, often in combination with other medicinal plant materials. The growing world market for skullcap, along with other medicinal herbs, has generated interest for its cultivation in Australia. Australia is agriculturally able to secure a share of the world market but various handling and quality issues need to be resolved for the nation to be successful at exporting and import substitution. The quality issues are being driven by consumers who are increasingly more demanding for consistent product quality, and as world crop supply starts to meet market demand there will be greater competition in product supply. Countries that can consistently supply high quality raw material and processed products will have preferential access to the premium market segment and thus maximise economic return.

The ultimate determinant of quality in all medicinal herbs, including skullcap, is the concentration of active constituents that impart a health benefit to consumers. While there is still some uncertainty on the relative effectiveness of various compounds, it is widely accepted that the flavonoid group of compounds are important active constituents in skullcap. The research studies described in this report used the total flavonoids as the marker of skullcap quality.

The aim of the project was to assist the Australian industry, that is, growers, traders and processors, to improve the quality of Australian-grown skullcap. The research objectives focused on determining:

• reliable methods for the analysis of flavonoids, • optimum plant sections and harvest times to maximise levels of flavonoids, • effect of postharvest handling practices on levels of flavonoids, • effect of solvent extraction on levels of flavonoids, and • levels of flavonoids in products available to consumers.

Efficient and reliable quantitative analytical methods for the analysis of flavonoids in skullcap were developed using high performance liquid chromatography (HPLC). The extraction method adopted was to clean by washing, dry in a heat pump dryer at 38°C, grind to a particle size of <200 µm, extract with 80% methanol/water at a solvent/solute ratio of 100:1, filter, wash the residue three times with 80% methanol, and make the extract up to 100 mL. Extracts were analysed by HPLC using a mobile phase of a linear gradient of 30%-90% methanol/water acidified with phosphoric acid, a stationary phase of a silica C18 column, and flavonoids peak detection by UV at 280 nm. Quantification of total flavonoids was based on the peak area of a working reference compound, chrysin, which was initially calibrated against a standard of baicalin. The same response factor was assumed to apply to all flavonoids. Values were determined on a dry weight basis with the water content of the dried sample determined by vacuum drying.

The HPLC separation required a total run time of 30 min and achieved optimal separation of 13 single peaks and 2 fused peaks. UV spectral analysis identified each peak as being a flavone, flavonol or flavanone. Mass spectroscopy was performed to attempt positive identification of the peaks but it was only possible to identify the major peak as baicalin with baicalin and scutellarin as minor components.

The flavonoids composition of skullcap plant parts was determined at the stage of maturity when plants are commercially harvested. The major compound in leaf, stem and root sections was baicalin at 40-50% of the total flavonoids. Peaks 5 and 6a were each present in leaf and stem at about 10% of the total while Peaks 8 and 8a were similarly present in the root. Thus, most of the flavonoids content was due to three compounds. The differences in the proportions of flavonoids in the plant sections would enable ready differentiation of leaf, stem and root sections based on comparison with their

characteristic chromatogram. The profile could be also used to detect the presence of adulterants that would alter the characteristic profile or amount of flavonoids present.

The concentration of total flavonoids in skullcap was determined in plants harvested at four growth stages from the stem formation stage to fruit set. The leaf section was found to have a higher flavonoids concentration than the other sections with the level in the root greater than in the stem. The leaf would therefore be the preferred plant section if end products of high quality were desired. Throughout the growing season, the concentration of flavonoids was highest in the leaf section when the leaves were immature. The root and stem sections did not show such a marked change with plant maturity although the root also tended to be highest in immature plants. Comparison of the flavonoids concentration between plant sections within a growth stage shows that the same trend occurs throughout growth of the level in leaf>root>stem. The total flavonoids present in each plant section, which reflects both the concentration of flavonoids and plant size, was found to be highest in plants at the commercial stage of harvest, which is just before fruit set. The effect was mostly due to a marked increase leaf weight during plant maturation that overcame the decrease in flavonoids concentration.

The trade in skullcap is primarily as dried aerial material with drying usually the responsibility of the grower. Since there is no published literature on the effect of postharvest operations on the active constituents of skullcap, examination was made of the effect of postharvest practices on the level of flavonoids in the final dried aerial plant material product. The need for careful handling after harvest was examined by inflicting controlled physical damage to plant material. Cutting the plant was found to not significantly affect the flavonoids level in the dried product but there was a substantial loss of flavonoids when plants were mechanically stressed. Thus, care must be taken in physical handling of fresh plants to maintain the flavonoids content.

The effect of a substantial time delay between harvest and drying on the retention of flavonoids was examined by holding freshly harvested aerial material at 20°C and 60% RH for 30 days. There was a rapid rate of evaporation of water in the first 3 days with equilibrium achieved by day 6 when about 70% of the original weight was lost. However, the plant material never reached the status of being commercially dry, which required another 10% of moisture to be lost. However, there was no significant decrease in the concentration of total flavonoids during storage for 30 days at 20°C. Thus, freshly harvested skullcap can be stored at 20°C and RH of 60% or less without any marked decrease in flavonoids. This would remove the necessity to dry plants immediately after harvest. Further study is required to determine the wider applicability of this finding, which at this stage can only be considered to apply to plants held under conditions where air is able to circulate around plants so that endogenous mould spores do not germination and natural drying of plants occurs at a relatively fast rate.

The effect of drying temperature was determined on harvested plants placed in a hot air drier until commercially dry. The time for plant material to commercially dry, that is, attain moisture content of 10%, was reduced as the temperature increased with drying time at 40°C about six times longer than at 70°C. There was, however, no decrease in total flavonoids concentration when skullcap was dried at temperatures from 40°C to 70°C. The results indicate that high temperature drying of skullcap is a feasible option as it is economically advantageous without any quality implications.

Dried skullcap is invariably stored for some time by various operators in the marketing chain. A study was undertaken to examine the changes in flavonoids in dried skullcap powder during storage for 60 days. Dried skullcap powder was spread on plates and placed at 5ºC and 20ºC in a chamber at low humidity, and in air of ambient humidity at 5°C, 20°C and 30°C in a darkened chamber and at 20°C under incandescent light. There was a decrease in total flavonoids concentration of about 0.05 mg/day during storage under all environmental conditions except for skullcap held at 5°C in ambient humidity where a higher rate of 0.85 mg/day occurred. The increased rate of loss at 5°C in ambient humidity was considered to be due to an increase in moisture content of the skullcap to about 30 g/100 g that triggered an increased enzymic activity. The moisture content of skullcap in all other treatments

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remained at ±4 g/100 g of the initial water content of about 7 g/100 g. The storage of dried ground skullcap at any temperature up to 30ºC, and protected from water uptake, thus resulted in a loss of about 0.1% of the flavonoids per day. This may be an important quality issue for long term storage. While this scenario is assumed to also apply for dried skullcap of a particle size greater than the 200 µm used in this study, the rate of loss for larger particle sizes would undoubtedly be lower and would need to be determined experimentally.

While traditional usage of skullcap was by herbal practitioners or individual informed citizens who utilised dried plant parts, most medicinal herbs in Western society are now marketed mainly as a range of solid and liquid manufactured products that may also contain other added ingredients. A common processing method is to extract dried plant material with a solvent of ethanol and water. The alcoholic extract is then either marketed as a liquid concentrate or dried and marketed as a tablet or capsule. The effect of extraction with 40-100% aqueous alcohol was examined with dried skullcap powder at room temperature at a solvent:solute ratio of 4:1. The proportion of total flavonoids extracted varied greatly with a maximum extraction of about 70% of the plant flavonoids obtained with 40-60% ethanol. Analysis of the herb residue remaining after extraction showed that the sum of the extracted and residual level of flavonoids was 70-80% of the original level, indicating degradation of 20-30% during extraction. Storage of the 40-60% ethanolic extracts at 20°C showed a 0.17% loss/day, which is about 50% greater than in dried skullcap powder. After 10 weeks storage, this rate of loss becomes about 12% and is probably of significance from a quality perspective in a marketed product. This may require adjusted formulation of the product to include flavonoid stabilisers in order to maintain an adequate shelf life.

A market survey identified 10 commercially available products labelled to contain S. lateriflora that were available from independent herb suppliers and health food stores. Four solid and one liquid product claimed to consist only of skullcap. The four dried skullcap products generated a flavonoids profile and concentration consistent with skullcap aerial parts analysed in the growth study of this project. The chromatogram of the liquid skullcap preparation, however, differed substantially including omission of baicalin, the major flavone found in this project, and was more consistent with a published profile for S. incana.

The other five products were mixed herbal tablet, capsule and liquid preparations. The analysis of these products may overestimate the skullcap content as some of the added herbs are known to contain flavonoids and they may not be fully separated from skullcap flavonoids in the analysis. Notwithstanding this limitation, no mixed liquid herbal preparation was found to contain a typical skullcap flavonoids profile with only one product containing more than 2 of the 17 flavonoids identified in this study. This suggests that considerable degradation of some flavonoids had occurred during processing and/or not all the skullcap added was S. lateriflora. A calculation of skullcap flavonoids activity in the products based on label claims suggest that one product contained the expected level of flavonoids, three contained 2-10% of the expected level while the other product could not be so evaluated as the label did not specify the skullcap content. The preparations with low medicinal quality must be of concern.

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1. INTRODUCTION

Scutellaria lateriflora L., with common names of skullcap, mad-dog weed, madweed, hoodwort, helmet flower, blue pimpernel and Quakers bonnet, is a plant indigenous to North America ranging from Canada to Florida and westward to British Columbia, Oregon and New Mexico. It is a perennial plant about 0.5 m high that inhabits the border of wet areas and flowers in the summer months resulting in characteristic helmet-shaped fruit (Millspaugh, 1974; Mills, 1994). Skullcap is commercially grown in Australia and New Zealand from seed and by root division. For the plant to grow well, moist soil and some shade are essential in dry summer weather as occurs in much of Australia. Skullcap struggles in direct open exposures but in Europe and the USA it grows successfully in direct sunshine (Hall, 1985).

The skullcap plant parts used medicinally are the aerial sections (leaf, stem and flower) although Hall (1985) states that the whole herb can be used in making medicinal tinctures and extracts. The plant is normally harvested just before the flower buds open but the time of harvest can vary from early spring when leaves are young and tender to late in the flowering period (Fisher and Painter, 1996) when the seed pods have appeared on the plant (Ody, 1993).

The Cherokee Native Americans utilised skullcap as an infusion and decoction. The infusion was made by steeping the leaves and flower sections in boiling water while the decoction utilised the roots and stems by simmering in boiling water. The infusion was taken to suppress menstruation and to stop diarrhoea while the decoction was used in the treatment of nerves, breast pain, and post delivery medication, and as a general kidney medicine. A mixture of moistened root and bear grease was employed as a dressing for sores, swelling, inflammations and other types of wounds (Hamel and Chiltoskey, 1975). The first mention of skullcap in American medical literature was by Dr van der Veer who in 1772 claimed it to be a curative and prophylactic in canine rabies. Subsequently, the medicinal uses of skullcap have been as a sedative, nervine, antispasmodic, and anticonvulsant (Millspaugh, 1974; Mills, 1994). No fatal toxicity has been associated with skullcap although large doses can cause dizziness, erratic pulse, mental confusion, twitchings of the limbs, and other symptoms indicative of epilepsy (Newall et al., 1996).

Skullcap is available to consumers in a number of forms including herbal teas, tablets, capsules and oral liquid preparations. In these preparations, skullcap is commonly combined with hops (Humulus lupulus), passionflower (Passiflora incarnata) and valerian (Valeriana officinalis) although many other plant materials are also used.

The chemical constituents of the Scutellaria genus include flavonoids, volatile oils, iridoids, diterpenoids, waxes and tannins (Wren, 1998) with most of the chemical interpretation within the genus based on S. baicalensis, S. indica, and S. ikonnikovii. Studies into the identification of flavonoids of S. lateriflora report baicalein and its glucuronide, baicalin as the major flavonoid present (Lehmann, R., Penman, K., Leach, D. and Waterman, P., Southern Cross University, pers. comm.). Scutellarin and scutellarein are cited in much of the practising herbalist literature as the major flavonoid (e.g. Fisher and Painter, 1996, Grieves and Leyel, 1998, Wren, 1998) although no supporting scientific literature was found with Lehmann et al. (2000) ascribing scutellarin as only a minor component of the flavonoids profile.

TLC and HPLC methods have been developed to create characteristic profiles for rapid identification of skullcap. The Therapeutic Goods Administration (TGA) of Australia has utilised both TLC and HPLC for fingerprinting multi-herb formulas including skullcap (Kelly and Nguyen, 1998). Skullcap is often substituted with other species of the Scutellaria genus (Mills, 1994). Scutellaria galericulata, a northern temperate zone (including Britain and Europe) plant, has recently been used as a substitute in the UK and Europe, but no such adulteration has been reported in the USA. Skullcap often dries to a low weight and is frequently substituted in the USA with the heavier and more abundant germander (Teucrium chamaedrys) (Keville, 1994) and with Teucrium canadense (Newall et al., 1996).

A major factor in the determination of quality in medicinal herbs is the concentration of those constituents that lead to a health benefit. At this stage, the flavonoids are the group of constituents that are considered to best monitor the quality of skullcap and were thus evaluated in this study. The overall aim was to identify practices that would enable the harvest of skullcap at maximum quality and to retain optimum quality during postharvest operations. This was pursued experimentally by determining:

• reliable methods for the analysis of flavonoids, • optimum plant sections and harvest times to maximise levels of flavonoids, • effect of postharvest handling practices of fresh material on levels of flavonoids, • effect of solvent extraction on levels of flavonoids, and • levels of flavonoids in products available to consumers.

2. ANALYSIS OF FLAVONOIDS

Flavonoids in various plant matrices have been successfully analysed by High Performance Liquid Chromatography (HPLC) over the past 20 years with the combination of reverse phase separation using gradient elution and sensitive detection of components by ultra-violet (UV) radiation allowing simultaneous qualitative and quantitative analysis. The TGA employs a linear gradient method for a skullcap extract in which both mobile phases are maintained at pH 2.6 using trifluoroacetic acid which gives resolution into seven peaks although the run time of 80 min is considered excessive (Kelly and Nguyen, 1998). Also, it is known that use of such a low pH will accelerate loss of column efficiency. The separation employed by West (1995) incorporated initial isocratic conditions followed by a linear gradient for 20 min. The resulting separation, whilst efficient with regards to time, was found to elute several fused peaks with only four being distinguishable. It was determined therefore that a more efficient HPLC method of analysis of skullcap extracts needed to be developed.

The method to extract skullcap flavonoids involved dried plant parts being ground to a particle size of <200 µm and extracted with 80% methanol/water at a solvent/solute ratio of 100:1. The mobile phase incorporated phosphoric acid to control pH, as it is known to optimise flavonoids separation, and a series of mobile phase gradients was investigated to optimize separation. Initial results using acetonitrile showed that the flavonoids eluted rapidly but with a number of fused peaks. It was decided to convert to methanol as the organic phase and increase the acidity of the aqueous phase. Flavone peak detection between 200-600 nm revealed 280 nm, which is within the principal (Band II) maxima for flavonoids (Markham, 1982), as the optimum wavelength to reduce interfering non- flavonoid spectra.

The adopted method was a linear gradient of 30%-90% methanol/water, acidified with 1% 0.001M phosphoric acid for water and 0.007M for methanol, on a silica C18 column which achieved optimal separation of 13 single peaks and only 2 fused peaks with a total run time of 30 min. The optimum separation method for each section can be seen in Figure 2.1.

Analysis of the chromophores by the characteristic absorption spectra of flavonoids in the ranges of 250-280 (Band II) and 310-350 nm (Band I) (Markham, 1982) identifies those peaks that were flavonoids and Table 2.1 gives details of the individual peaks. Peaks 6 and 9 correspond with spectra obtained for standard compounds of baicalin and baicalein, respectively, as seen in Figure 2.2. Peaks 3, 4, 6a, 7b, 8a and 10a correspond with the class of flavones as the wavelengths are within the absorption range of both Band I and II. Peaks 1, 7, 8 and 10 show indicate a flavanone structure by the characteristic shoulder at 300-330 nm. Peak 5 corresponds to the characteristics of a 3-OH substituted flavonol (Markham, 1982). The analysis was thus able to distinguish between flavanones and other compounds found in skullcap that absorbed at the same wavelength.

Mass spectroscopy was performed to attempt positive identification of the major peaks in the skullcap analysis. Mass spectral data for the major peaks generated from leaf, stem and root material are given in Table 2.2. The major peak found in the leaf, stem and root sections was positively identified as baicalin. This confirms the findings of West (1995) and Lehmann et. al.(2000) and refutes suggestions that scutellarin is the major flavonoid. Common fragments of flavonoids include 65-69, 77, 79 and 91 m/z. The 168 m/z fragment is the main signal in peak 2 while 270 m/z is the main signal for peak 1, the latter corresponding to that of standard baicalin.

From the 17 peaks found in HPLC analysis, the electron impact method of mass spectroscopy was found to be unsatisfactory for flavonoid analysis with only three peaks being distinguishable within the total ion chromatogram. Interpretation of the data revealed that the major peak found in the leaf, stem and root sections was positively identified as baicalin with baicalein and scutellarin as minor components.

For routine analysis, quantitation of the resolved peaks was based on the peak area of a working reference compound, chrysin (Sigma-Aldrich, Castle Hill, NSW) that was used as an external standard. Chrysin was initially calibrated against baicalin and the same response factor was used for all flavonoids assuming a similar extinction coefficient all flavonoids. Some pharmacological studies have been conducted on individual flavonoids present in skullcap, but since the biological activity of all individual flavonoids is not available, the concentration of total flavonoids seemed the most appropriate measure of medicinal quality. All values are reported on a dry weight basis with the water content of the ground powder determined by drying in a vacuum oven for 12 hr at 100°C.

Table 2.1 Chromophore analysis of skullcap peaks generated by HPLC.

Peak Band II Band I Description Flavonoid No. 250-280 310-350 Type 1 286 - Major single peak, shoulder at 330 nm. Flavanone 2 - 315 Major single peak, no apparent shoulder None 3 284 335 Two peaks, no apparent shoulder Flavone 3a 4 273 335 Two peaks, no apparent shoulder Flavone Two peaks, major at Band II, minor at Band I 3-OH Sub 5 288 360 Flavonol 6 277 315 Two peaks, no apparent shoulder Flavone 6a 275 331 Two peaks, no apparent shoulder Flavone 7 281 - Major peak with small shoulder at 330 nm Flavanone 7a 7b 273 310 Two peaks, no apparent shoulder Flavone 8 273 - Major peak with large shoulder at 345-350 nm. Flavanone 8a 271 329 Two peaks, one large and one small broad Flavone 9 277 323 Two peaks, no apparent shoulder Flavone 10 275 - Major peak, large shoulder at 345-350 nm. Flavanone 10a 271 319 Two peaks, no apparent shoulder Flavone

Figure 2.1 Separation of flavonoids from a) leaf, b) stem and c) root of skullcap plants.

a) Baicalin & Peak 6, Stage 4 Leaf

b) Baicalein & Peak 9, Stage 4 Leaf

Figure. 2.2. Chromophore comparison of (a) peak 6 and baicalin standard, and (b) peak 9 and baicalein standard.

Table 2.2 Mass of major spectral fragments obtained from skullcap plant sections at growth Stage 4 and reference standards.

Sample Ret. time Fraction mass (min) 18 51 65 67 69 77 79 91 102 139 140 168 242 270 284 286 Baicalin* 18.2 √ √ √ √ √ Baicalein* 22.8 √ √ √ √ √ √ √ √ Scutellarein* 16.2 √ √ √ √ Leaf 1 18.1 √ √ √ √ √ Leaf 2 19.4 √ √ √ √ Stem 1 18.3 √ √ √ √ √ Stem 2 19.4 √ √ √ √ Root 1 18.1 √ √ √ √ √ Root 2 19.4 √ √ √ √ Root 3 21.3 √ √ √ √ √ √

* Standard from Apin Chemical (Oxon, UK)

3. FLAVONOIDS IN SKULLCAP DURING GROWTH

Skullcap seeds and seedlings obtained from Subiaco Herb Farm (Walcha, NSW) were grown at Ourimbah, NSW with plants receiving morning sun only and the soil was kept moist at all times. Whole plants were harvested at four different growth stages: 1 = fibrous root, small ovate leaves and initial stem formation; 2 = fibrous root, medium ovate leaves and distinct square stem, buds forming; 3 = fibrous root, medium ovate leaves, square stem, raceme and small blue flowers formed; and 4 = fibrous root, medium ovate leaves, square stem and fruiting calyx formed. Plants were then separated into root and aerial parts for Stage 1 and root, leaf and stem for stages 2, 3 and 4. The Stage 3 leaf section included flowers while the Stage 4 leaf section included fruit.

Harvested plants were gently rinsed with water and excess foreign material removed before being dried in a heat pump dryer at 38°C. The drying time for Stage 1 plants was 12 hr, for Stage 2 was 24 hr and 36 hr for Stages 3 and 4. Dried samples were analysed for flavonoids.

3.1 Flavonoids composition

The data in Table 3.1 show the proportion of individual flavonoid peaks in the sections of plants at full maturity (Stage 3), which is when plants are commercially harvested. The major compound in all sections was baicalin (Peak 6), which was present at 40-50% of the total flavonoids. The other major compounds present were Peaks 5 and 6a which were each present in leaf and stem at about 10% of the total flavonoids while Peaks 8 and 8a were similarly present in the root. Thus, most of the flavonoids content was due to a relatively small number of compounds. Even though other peaks were relatively small, there were statistically significant differences in the proportions of many peaks in the different plant sections. This means that the separate plant sections have a characteristic chromatographic profile that could be used to identify the plant section used in a product. It could also be a useful tool to detect the presence of an adulterant in a sample, as it would alter the flavonoids profile or total content.

Table 3.1 Proportion of peaks of skullcap plant sections at commercial maturity (Stage 3).

Peak no. % of Total peak area Leaf Stem Root Signif. diff. 1 0.5 0.7 1.8 ** 2 1.2 0.5 0.4 ** 3 2.3 1.7 0.7 *** 3a 0.9 0.9 1.0 ns 4 5.9 4.2 3.2 ** 5 10.2 8.0 5.4 *** 6 54.5 45.2 38.1 ** 6a 13.7 12.8 6.8 *** 7 1.3 2.5 2.6 ** 7a 0.0 2.4 3.4 *** 7b 4.5 6.6 4.0 ** 8 1.6 5.1 12.4 *** 8a 0.9 4.4 10.3 *** 9 1.7 2.2 3.4 ns 10 0.1 1.3 5.8 *** 10a 0.2 1.5 0.9 ** TOTAL 100 100 100 ns (not significant at P=0.05), *(P<0.05), **(P<0.01), ***(P<0.001)

3.2 Level of flavonoids in plant sections The concentration of flavonoids present in each plant section averaged across the four stages of plant maturity is shown in Table 3.2. The leaf section contained a significantly higher flavonoids concentration than the other sections with the level in the root greater than in the stem. The leaf would therefore be the preferred plant section if end products of high quality were desired.

Table 3.2 Mean flavonoids concentration of skullcap leaf, stem and root plant sections across the growing season.

Flavonoids concentration (mg/g) Leaf Stem Root LSD (5%) 50.8 21.3 35.8 ±3.0

LSD is the least significant difference at P=0.05

The concentration of flavonoids in each plant section throughout a season of growth is given in Table 3.3. It is seen that the flavonoids were highest in the leaf section at Stage 1 when the plants were immature. The root and stem did not show such a marked change with plant maturity although the root also tended to be highest at Stage 1. Comparison of the flavonoids concentration between plant sections within a growth stage shows that the same trend occurred at each Stage with the level in leaf>root> stem.

This is the first reported study on the flavonoids content of S. lateriflora during growth but Zhang et al.(1998) reported that the flavonoids content in the roots of S. baicalensis were highest at the end of summer (probably Stage 3) and Wang et al. (1990) reported that the level in S. ikonnikovii was greatest in mid-summer for aerial sections.

Table 3.3 Flavonoids concentration in skullcap plant sections at four growth stages in a season.

Plant Flavonoids concentration (mg/g) section Stage 1 Stage 2 Stage 3 Stage 4 LSD (5%) Leaf 69.3 47.7 52.9 44.4 ±5.3 Stem ~ 21.3 22.9 21.0 ±2.0 Root 40.3 34.5 32.4 37.6 ±4.4 LSD (5%) ±5.7 ±4.7 ±6.0 ±4.1

The total flavonoids present in each plant section, which reflects both the concentration of flavonoids and plant size is given in Table 3.4. The data show that plants at Stage 3 contain the highest amount of flavonoids. The effect was mostly due to a marked increase leaf weight during plant maturation that overcame the decrease in flavonoids concentration noted in Table 3.3. The total flavonoids content was not determined on the root section as the matted structure of the root makes it difficult to estimate the weight of root that should be attributed to an individual aerial section.

Table 3.4 Total flavonoids content of skullcap plant sections during growth.

Plant Flavonoids content (mg/plant section) section Stage 1 Stage 2 Stage 3 Stage 4 LSD (5%) Leaf 17.4 20.1 45.0 36.5 ±4.2 Stem 2.9 9.9 9.1 ±0.85

4. POSTHARVEST HANDLING AND DRYING

The current trade in skullcap is primarily as dried aerial material rather than as fresh plant. The drying of skullcap is invariably the responsibility of the grower who may dry the crop on farm or sub-contract drying to another grower or group. It needs to be recognised that a ‘dried’ skullcap product still contains a measurable amount of water. While there is no set standard for the moisture content of dried skullcap, levels found in dried raw material ranged from 5-10 g/100 g. The water content of fresh aerial material is about 75 to 80g/100 g. The hygroscopic nature of dried skullcap is not well documented and it was noted during the study that the equilibrium moisture content varied depending on the ambient relative humidity and particle size of the dried material.

As the scale of production on a farm increases or a larger off-farm drier is utilised, there is increasing handling and transport of the freshly harvested crop and increasing delay between harvest and drying. For the wholesaler and manufacturer, there is increasing storage of the dried skullcap and increasing delay between receipt of the crop and processing into a manufactured product. These constraints are due to the seasonal nature of the skullcap harvest and the economic desirability of operating processing plants throughout as much of the year as possible.

There is no published literature on the effect of postharvest operations on the active constituents of skullcap. A series of studies was therefore conducted to examine the effect of various postharvest practices on the level of flavonoids in the final dried product of aerial plant material.

4.1 Physical damage to freshly harvested plants Even when skullcap is harvested and dried on farm with little delay before drying, the crop is subject to a range of handling operations arising from the harvest operation itself, transporting from the field to the drier in bags or in bulk, and in pre-drying operations such as cleaning, sorting, separating plant parts and forcing plants to fit onto drying trays. All of these operations can inflict physical damage on the fresh plant with the resultant disruption of plant cells and structures. Since the fresh plant is still metabolically active, such damage could result in enzymic or chemical changes to the flavonoids.

Mature skullcap plants from the Central Coast of NSW were harvested and subjected to various manual treatments designed to inflict varying degrees of physical damage to the plant cells and structure. The treatments designated as D0 to D3, were:

• D0: careful handling to inflict minimal damage, • D1: cut into 1cm2 sections, • D2: cut into 1cm2 sections and compressed for leaves to be visibly crushed, • D3: treated as in D2 but with 24 hr time delay before drying.

After treatment, plants were placed in a hot air drier at 40°C for 24 hr to dry the samples.

The level of flavonoids in the dried samples is given in Table 4.1. This shows that there was no significant effect on the flavonoids level in dried material cut into sections. However, there was a significant loss of flavonoids when plants were mechanically stressed. Thus, care must be taken in physical handling of fresh plants to maintain the flavonoids content.

Table 4.1. Effect of physical damage on retention of flavonoids during drying of skullcap.

Damage level D0 D1 D2 D3 LSD Flavonoids (mg/g) 53.5 57.8 40.2 40.1 ±5.3

4.2 Delay between harvest and drying The effect of a substantial time delay between harvest and drying on the retention of flavonoids was examined on freshly harvested aerial material. Plant material was stored at 20°C and 60% RH and the moisture content was determined at intervals up to 30 days. The data in Table 4.2 show that there was a rapid rate of evaporation of water during storage in the first 3 days with equilibrium achieved by day 6 when about 70% of the original weight was lost. However, the plant material never reached the status of being commercially dry which required another 10% of moisture to be lost. Table 4.2 also shows that there was no significant decrease in the concentration of total flavonoids during storage for 30 days at 20°C.

Fresh skullcap can therefore be stored at 20°C and an RH of 60% or less without any decrease in the level of flavonoids. This removes the necessity to dry plants immediately after harvest. The caveat in the results is that the findings at this stage can only be considered to apply to plants held under conditions where air is able to circulate around plants so that germination of endogenous mould spores does not occur and natural drying of plants occurs at a relatively fast rate. This would mean holding plants on mesh trays rather than in sacks or bins. While it may be shown in a subsequent study that holding fresh plants in an enclosed environment may not greatly affect the level of flavonoids, the risk of mould growth would be an important consideration.

Table 4.2. Weight loss and flavonoids concentration of skullcap during storage at 20°C and 60% RH.

Day 0 3 6 10 13 20 30 Weight loss (g/100 g) during storage 0 67 71 72 71 72 71 Flavonoids concentration (mg/g) 53.5 54.7 61.7 53.2 LSD ±16.0

4.3 Drying temperature An examination was made of changes in total flavonoids in fresh skullcap subjected to hot air drying at a range of air temperatures. Harvested plants were placed in a hot air drier until commercially dry. In order to determine the drying time required at each temperature, a preliminary study was conducted where plants were weighed at periodic intervals and the time to reach 80% weight loss was taken as the time needed to dry the sample.

The data in Table 4.3 show that the time for plant material to attain moisture content of 10% was reduced as the temperature was increased with the drying time about six times longer at 40°C than at 70°C. There was, however, no significant difference in total flavonoids concentration when skullcap was dried at temperatures from 40°C to 70°C. The results thus indicate that high temperature drying is a feasible option as it is economically advantageous without any quality implications.

Table 4.3. Time for skullcap held in a hot air drier to become commercially dry and the flavonoids concentration in dry aerial material.

Drying temp. Flavonoids conc. (mg/g) Time to dry (hr) 70°C 48.1 1 50°C 44.4 2 40°C 51.5 6 LSD ±7.70

4.4 Storage of dried material Dried skullcap is invariably stored for some time by various agents in the marketing chain before it is consumed or processed. A study was undertaken to examine any change in flavonoid concentration in dried skullcap powder during storage for 60 days. The powder was evenly spread on a Petri dish where the contents were highly exposed to the atmosphere. Ground skullcap was used, as it would be more reactive to environmental conditions than intact plant parts and thus, degradation trends would be more easily observed. The dishes were placed at 5°C, 20°C and 30°C in a darkened chamber at ambient humidity (designated as ‘moist air’), and at 20°C under incandescent light bulbs. Two additional samples were placed at 5ºC and 20ºC but were protected from atmospheric moisture in a desiccator with silica gel to absorb any moisture (designated as ‘dry air’).

Figure 4.1 shows that there was a slight decrease in total flavonoids concentration in skullcap during storage under all environmental conditions with a much higher loss at 5°C in high humidity than in the other storage environments. A linear regression y=a+bx (where y is flavonoids concentration, and x is time in days) was fitted for all treatments. The slope of the equations, that is, the rate of loss of flavonoids ranged from 0.03-0.07 mg/day with no significant difference between treatments, except for skullcap held at 5ºC in ambient humidity where a significantly higher rate of 0.85 mg/day occurred.

Figure 4.1. Change in flavonoids concentration of dried skullcap powder stored in various environmental conditions for 60 days.

20 Concentration (mg/g)

0 0204060 Time (days)

The water content of the samples was also determined at each sampling time in order to allow the flavonoids data to be presented on a dry matter basis. Examination of these data showed that the initial water content of about 7 g/100 g was maintained at ±4 g/100 g during storage in all treatments except for skullcap stored at 5°C in an atmosphere that was cooled ambient air and therefore of quite high humidity, which showed an increase in water content to about 30 g/100 g. The enhanced degradation of flavonoids at 5ºC in moist air was undoubtedly due to increased enzymic activity arising from an increased water content.

The finding that loss of flavonoids was not directly related to temperature indicates that dried ground skullcap stored at any temperature up to 30ºC will lose about 0.1% of flavonoids per day. Thus, storage period is an issue for maintenance of quality and possibly should not extend beyond 100 days when a loss of 10% of the flavonoids will have occurred. The data also emphasise the importance of protecting dried skullcap from moisture uptake whatever the storage temperature. It is assumed that the loss of flavonoids during storage also holds for dried skullcap of a particle size greater than the 200 µm used in this study, but the rate of loss for such larger particle sizes would undoubtedly be slower and would need to be determined experimentally.

5. EXTRACTION AND PRODUCT QUALITY

Traditional usage of skullcap, in common with other medicinal herbs, was by natural medicine practitioners or individual informed citizens who utilised dried plant parts by direct consumption or through production of a range of infusions or poultices. However, use of most medicinal herbs in Western society is now mainly through a range of solid and liquid manufactured products such as tablets, capsules and liquid extracts that may also contain other added ingredients. These products are increasingly generated by large manufacturers using standard pharmaceutical or food processing techniques and made available to alternative therapists or directly to consumers through pharmacies, supermarkets and alternative lifestyle outlets.

A common processing method for the manufacture of skullcap products in Australia is by initially obtaining an extract from dried plant material with a solvent containing a mixture of ethanol and water. The alcoholic extract is then either marketed as a liquid, with possible adjustment of the alcohol content, or spray dried and marketed, with added solid filler, as a tablet or capsule.

5.1 Effect of ethanol:water ratios There is no published literature on the effect of processing operations in the extraction of active constituents of skullcap. This study examined the effect of alcoholic solvent mixtures in extracting the total flavonoids from skullcap aerial material.

Dried skullcap powder was extracted at room temperature (about 20°C) with ethanol:water mixtures containing 40-100% ethanol at a solvent:solute ratio of 4:1. The data in Table 5.1 show that the proportion of flavonoids in skullcap that were extracted varied greatly with the concentration of ethanol in the solvent. The maximum amount extracted from aerial material was about 70% and was obtained with 40-60% ethanol. The extraction level decreased with increasing alcohol content and <1% was extracted with 100% ethanol.

To determine the amount of flavonoids available for extraction and how much was being degraded by the extraction process, analysis was performed on the herb residue remaining after extraction using 80% methanol, which is known to completely extract the flavonoids. The data in Table 5.1 show that the sum of the extracted and residual level of flavonoids was 70-80% of the original level when 40-80% ethanol was used for extraction, indicating degradation of 20-30% during extraction and a high level of extraction of remaining flavonoids. There was considerable degradation of flavonoids with 100% ethanol.

Table 5.1. Effect of ethanol:water solvent ratio on extraction of flavonoids from skullcap powder.

Solvent, % of Original flavonoids % ethanol Extract Residue Total 100 <1 43 44 80 56 14 70 60 73 6 78 40 72 2 75 LSD ±10.2 ±5.1 ±10.5

The stability of flavonoids in the ethanolic extracts during storage at 20°C for 10 weeks was examined to gauge the possible shelf-life of commercial extracts. The data in Table 5.2 show that extracts in 60 and 40% ethanol resulted in a 11-13% loss in flavonoids during the 10 weeks of storage, an average loss of 0.17 %/day. While the flavonoids were more stable in these solvents than in 80% ethanol, the loss would seem to be significant from a commercial quality perspective and may require adjusted formulation to include flavone stabilisers to maintain shelf life of a product.

Table 5.2. Loss of flavonoids in skullcap extracts during storage at 20°C for 10 weeks.

% Ethanol % Loss of extracted flavonoids 80 60 60 11 40 13 LSD ±9.2

5.2 Analysis of commercial products A total of 10 commercially available products labelled to contain S. lateriflora were purchased from independent herb suppliers and health food stores. The purchase consisted of four solid and one liquid products claimed to consist only of skullcap. The other five were mixed herbal preparations that included skullcap and were tablet, capsule and liquid products. The additional material and known active ingredients stated to be in these products are given in Table 5.3.

Analysis of products containing a mixed source of compounds required identification of only those flavonoids that matched the retention time and UV chromophore of skullcap. All other instrumental responses were ascribed as originating from accompanying herbal material. It is recognised that some overestimations of skullcap flavonoids may occur as some of the added herbs such chamomile, hops, passionflower, peppermint and strawberry do contain flavonoids (Wren, 1998) and may not all be separated from skullcap flavonoids in the analysis.

Table 5.3 Processed herb products containing skullcap labelled as Scutellaria lateriflora.

Brand Type Origin Herbal ingredients (as per label) Austral Seeds & Powder Australia Skullcap Herbs Mediherb Powder USA Skullcap Mediherb Powder USA Skullcap Mediherb Powder USA Skullcap Southern Cross Liquid Australia Skullcap Herbal Chamomile, passionflower, valerian, catnip, skullcap, Red Seal Tea NZ strawberry leaves, peppermint Sleep Ezy Tablet Australia Valerian, skullcap, hops, passionflower Silent Night Capsule USA Hops, valerian, skullcap Jamaican dogwood, valerian, Corydalis ambigua, Sleep Easy Liquid Australia passionflower, skullcap Chamomile, hops, passionflower, valerian, mistletoe, Restful Sleep Liquid Australia skullcap, white chestnut

Table 5.4 Flavonoids in commercially available processed products containing skullcap.

Product Flavone concentration (mg/g) 1* 2 3 3a 4 5 6 6a 7 7a 7b 8 8a 9 10 10a Total Austral 0.3 0.7 1.3 0.4 2.1 3.9 17.6 3.8 0.5 0.2 1.7 0.9 0.5 0.8 0.2 0.3 35.1 MediHerb1 0.5 0.7 1.2 0.3 0.7 6.6 25.5 1.4 0.9 0.9 2.0 1.2 0.7 1.6 0.3 0.2 44.7 MediHerb2 0.4 0.8 1.9 0.3 2.5 4.6 25.0 3.5 0.8 0 2.4 0.9 0.6 1.4 0.3 0.6 46.1 MediHerb3 0.5 0.9 2.2 0.4 2.9 5.2 28.0 4.1 0.9 0 2.6 0.8 0.5 1.2 0.2 0.5 50.9 Southern Cross 0 0 3.9 0 0 0 0 0 0 0 0 0 0 0 1.5 0 5.4 Red Seal Tea 0 0 <0.1 0 0 0 0.4 0 0 0 0 0 0 0 0 0 0.4 Sleep Ezy 0 0 0 0 0 0 0.2 0 0 0 0 0 0 0 0 0 0.2 Silent Night 0 0 <0.1 0 0 0 0.3 0 0 0 0 <0.1 0 0 0 0 0.4 Sleep Easy 0 0 <0.1 0 <0.1 0 0.6 0.2 0 0 0 0 0 0 0 0 0.9 Restful Sleep 0 0 0 0 0 0 0.1 0 0 0 0 0 0 0 0 0 0.1 * Numbers are the peaks obtained by HPLC as given in Table 2.2.

The three Mediherb dried herb samples from USA origin material produced a flavonoid profile and concentration matching the profile and quantity of skullcap aerial parts found in the growth study of this project. The Austral sample from Tasmania was observed to have fruit from the plant included, indicating harvesting occurred within growth Stage 4 and it was found to have similar flavone content to Stage 4 aerial parts. The chromatogram of the liquid skullcap preparation was, however, quite different with the most notable difference being the absence of biacalin which was the major flavone constituent found in this project. The chromatogram of S. incana published in the TGA herbal screening literature (Kelly and Nguyen 1998) revealed a close match and it is suggested to be the source material rather than S. lateriflora although analysis of additional samples is needed for this be a firm conclusion.

None of the mixed liquid herbal preparations contained all of the flavonoid constituents of skullcap found in this project and suggests that considerable degradation of some constituents has occurred during processing and/or not all the skullcap was added as S. lateriflora. A calculation of skullcap flavonoids activity in the products was made by allowing for dilution based on the label stated amount of skullcap per unit of product, the measured concentration of total flavonoids and an average flavonoids concentration in dried skullcap of 50 mg/g. Firstly, it is of note that many of these preparations had as few as 2-3 of the 17 flavonoids observed in dried fresh skullcap. Secondly, the results in Table 5.5 show the amount of flavonoids expected in products when calculated from average flavonoids concentrations obtained in the growth study and that actually found in the products. One product contained the expected level of flavonoids, three products contained 2-10% of the expected level, and the other product could not be evaluated, as the label did not indicate the amount of skullcap in the product.

Table 5.5 Total flavonoids in herbal preparations.

Product Skullcap Flavonoids (mg/g) % label amount Expected* Analysed Retained Red Seal tea ns/1.5 g sachet ns 0.43 ? Sleep Ezy 100 mg/ 1 g tablet 5 0.15 3 Silent Night 127 mg/0.38 g capsule 16.7 0.35 2 Sleep Easy 83.5 mg/5 mL 0.8 0.90 108 Restful Sleep 12.5 mg/mL 0.6 0.07 11 ns, skullcap amount not stated *Assuming a flavonoids concentration of 50 mg/g plant materials as there was no label claim.

6. DISCUSSION AND RECOMMENDATIONS

6.1 Need for analysis of flavonoids The various Scutellaria species used in medicinal herb preparations and the increasing consumer interest in the quality of complementary health products has created a need to correctly identify the species in a particular preparation and to quantify the health efficacy of each ingredient. The added urgency for having such methodology for S. lateriflora stems from the paucity of analytical data that currently exist with some uncertainty as to the major flavonoids in the species. The HPLC method developed in this study was found to be efficient for both the separation and quantification of skullcap flavonoids generating 17 eluted peaks with only two being fused peaks with a total run time of 30 min. The method was able to not only differentiate between Scutellaria species but also between individual plant sections of S. lateriflora. This therefore means a tool is now available to industry to rapidly and reliably assess the quality of raw material being traded and manufactured products generated from this skullcap. The skullcap plants analysed showed that baicalin is the major flavone present at about 2.5% dry weight and represents 40-50% of the total flavonoids.

6.2 Variation in flavonoids between plant sections The plant growth study highlighted large differences in the total flavonoids in leaf and stem material with the concentration in leaves about twice that in stems, and the total amount in plant leaf about four times that in total plant stem. Plant material that maximises the leaf to stem ratio will therefore have a higher concentration of total flavonoids. If industry is interested in raising the medicinal quality of traded skullcap plant material, this could be achieved by minimising the amount of stem in a bulk sample. There would, however, need to be a price differential based on quality for growers to have the incentive to selectively segregate leaf and stem material. It is worth noting that skullcap root contains flavonoids at a concentration considerably higher than stem but lower than leaf. The root is therefore a higher quality plant section than the stem for yield of flavonoids. It is not known whether the industry is currently using the root within harvested material, as growers may prefer to allow the root material to regenerate for the subsequent year. If the root is being discarded, consideration needs to be given to its utilisation by industry. However, it was found that the flavonoids composition varies substantially between leaf, stem and root plant sections. At this stage, the relative health efficacy of individual flavonoids is not known, but later elucidation could influence the desirability of the various plant sections.

The current method of harvesting plants at flowering maturity has been confirmed as the optimum time to maximise the flavonoids content. It should be noted, however, that if harvesting is required to be extended over a longer period, it is preferable to harvest from flowering through to fruit formation rather than at more immature stages.

6.3 Need for improved postharvest handling practices The finding that undamaged fresh skullcap plants can be held for extended periods at 20°C with little decrease in the level of flavonoids has considerable infrastructure and operational implications for industry. The ability to hold plants on farm before drying can reduce the size of a drier that needs to be installed with resultant substantial cost savings. In addition, the natural drying by ambient air that occurs during such delay will allow plants to be partially dried before being placed in a drier and hence generate an energy saving. An offsetting consideration is the need for increased storage racks and shed space to spread the plants during the holding period. If plants are stored in bins or sacks without adequate ventilation, there is a considerable danger of mould growth. The findings that aerial plant material was within 10% of being commercially dry after only 6 days raises natural air drying as a possibility in climates where low humidity conditions prevail during the harvest period and shed storage space is not a limitation. However, further investigation is required before such a radical step should be commercially implemented.

The limited study on drying indicates that hot air drying is a suitable method for skullcap, as even use of temperature of 70°C did not result in any noticeable loss of flavonoids in the dried product. The use of such a high temperature is recognised to cause loss of active constituents in other medicinal herbs. At 70°C, the drying time is substantially reduced than at lower temperatures thus increasing throughput. With other medicinal herbs, other drying technologies such as heat pump are recommended to increase plant throughput while using a lower temperature but these driers are more expensive to purchase and would seem to not offer any benefit in the drying of skullcap.

Storage of dried skullcap powder at temperatures up to 30°C resulted in a 0.1% loss of flavonoids per day and will have accumulated to a 10% loss after about 3 months. Losses in short term storage can therefore be considered acceptable but industry needs to be mindful that loss is continually occurring. However, if the skullcap is stored under conditions that allow absorption of moisture by the dried skullcap, a more rapid loss of flavonoids will occur. This means either holding the skullcap in air of low humidity, or in a package that minimises contact with ambient air. The studies were only on ground skullcap and thus only have direct application for processors. The rate of degradation of dried, non-ground skullcap would undoubtedly be at a slower rate but this needs further evaluation.

6.4 Need for improved processing operations The processing study while limited, showed that the flavonoids were most efficiently extracted from skullcap powder with 40-60% aqueous ethanol with about 70% of flavonoids passing into the solvent. The remaining 30% were mostly degraded during the extraction process as little flavonoids remained in the herb residue. A loss of 30% must be considered of some concern and further studies should examine a wider range of processing parameters such as solvent temperature, mode of extraction, and ratio of solvent to solute to attempt an increase in amount of extracted flavonoids. It was rather surprising that use of 100% ethanol did not result in the extraction of flavonoids to any extent with >50% degraded during the extraction process. Storage of the ethanolic extracts under ambient conditions resulted in the loss of 0.17% of the extracted flavonoids per day. This rate of loss is greater by about 50% than for dried skullcap powder and therefore is a more important consideration. If the extract is held for a few weeks by the manufacturer before being transformed into a dry product the loss may be acceptable but if the ethanolic extract is the marketed product, shelf life needs to be much longer than 10 weeks and the loss can be substantial in the hands of the consumer. Further investigation is required to minimise the rate of degradation during storage, possibly by the addition of some protective agents or the use of low temperature storage. Data are also required to understand the degradation mechanism and could assist in elucidating more efficient storage methods. The instability of flavonoids in the higher ethanolic solutions also warrants investigation.

The data on extraction and of solution stability could explain the variable retention of flavonoids in liquid and solid retail preparations. While only a limited number of manufactured herbal preparations were available for analysis, many showed a total flavonoids content much lower than would be expected from the label claims of the amount of skullcap incorporated into the products. Such a discrepancy in the level of active ingredients must be of concern to the industry for the efficacy of its products and can only fuel the disquiet felt by various groups in the community. The targeting of manufacturing for the discrepancy is supported from the analysis of products containing only dried skullcap where the flavonoids were at expected levels. The other concern from analysis of manufactured products was whether the added skullcap was always S. lateriflora, which is an equally serious issue for industry.

7. BIBLIOGRAPHY

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