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Generation of High Quality Australian Skullcap Products
by R.B.H. Wills and D.L. StuartMarch 2004
RIRDC Publication No 04/020 RIRDC Project No UNC-6A
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 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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