Objective 3. Improved agronomic practices for milk thistle.

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Milk Thistle Background

Milk Thistle Germplasm

New Lines Released by the University of Saskatchewan

Milk Thistle Agronomy

Seeding Rate and Desiccation Method Trials for Milk Thistle

Milk Thistle Value Added

Grading Milk Thistle

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Milk Thistle Background

Milk thistle (Silybum marianum (L.) is a flowering plant belonging to the daisy family (Asteraceae).  Milk thistle is known by several other names, including Blessed Milk thistle, Spotted thistle, St. Mary's thistle, Marian thistle, Holy thistle and Variegated thistle. Milk thistle is native to the Mediterranean regions of Europe, North Africa and the Middle East but has been introduced throughout the world. The plant is valued for its medicinal properties, but it is sometimes grown as an ornamental because of its unusual leaves.  Milk thistle spreads quickly and is considered a weed in some parts of the world.  In the U.S.A., it grows wild in most southern states and the north-eastern and mid-west states. It has been declared a noxious weed in Washington, Oregon and Texas, but is not considered as a noxious weed in any Canadian province.  

Plant Description:

Milk thistle is a vigorous tall upright plant that prefers dry sunny conditions.  The spiny stems branch at the top, and reach a height of approximately 1.2 to 2 m. The spiny leaves are wide, with white blotches or veins. Milk thistle gets its name from the thistle like appearance of the leaves and the milky white sap that exudes from any cut surface.  Flowers are generally red-purple.  A solitary flower develops at the end of each stem.  Milk thistle has an indeterminate growth and flowering habit, resulting in uneven development and maturity of flower heads.  The mature fruit (achene) is relatively small with an attached white silky pappus.  In the literature, the achenes, i.e. the ‘fruit’ are mostly (wrongly) referred to as ‘seed’. The immature fruit is soft and cream, tan, or brown in colour and the mature fruit is hard-skinned, shiny brown or brown with tan spots.  Due to the indeterminent flowering habit at any point in time the milk thistle plant has some flowers that are still opening while others have progressed to the point where the seed is shattering.  To address this issue of uneven seed maturity the mature heads are usually gathered by selective hand harvest.  The large thorns on the stems, leaves and seed heads of milk thistle make hand harvest of the seed heads an exceedingly unpleasant task.   While once-over machine harvest is clearly preferable from the perspective of efficiency, seed yield and quality may be compromised.  

 In the relatively cool and short Saskatchewan growing conditions, milk thistle is grown as an annual while in warmer longer growing environments milk thistle can be grown as a biennial.

Uses of Milk Thistle:
Every part of milk thistle including the stems, leaves, flower buds are edible. Milk thistle has been used for over 2,000 years as a herbal remedy for a variety of ailments, particularly for liver and gall bladder problems.  Studies suggest that substances in milk thistle (especially a flavonoid called silymarin) protect the liver from toxins, including certain drugs such as acetaminophen (Tylenol), which can cause liver damage in high doses. Silymarin has antioxidant and anti-inflammatory properties, and it may help the liver repair itself by growing new cells.

The leaves are used as a salad green or cooked.  Leaves can be trimmed of prickles and boiled or added raw to salads.  There are as yet no known medicinal properties for milk thistle leaves.  It is claimed that in recent times a number of herbal medicine manufacturers have introduced products containing milk thistle leaves but these products had no apparent therapeutic value. 

Constituents:

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Milk Thistle Germplasm Trials in 2005

Methodology

Lines tested in 2005 were; a) adapted lines tested in 2004 and b) any additional lines received. 

 The field test was conducted at the Department of Plant Sciences, Horticulture Field Research Station in Saskatoon.  This site was found to be well suited to production of milk thistle - based on the 2004 trial results.           

The trial site was prepared by disking, then rotovating two weeks prior to seeding. As a function of heavy fertilizer applications in previous crops, fertility levels in the test area were > 100 kg N/ha and > 120 kg P2O5/ha.  These fertility levels would easily meet the needs of most herbaceous medicinals and may actually be somewhat excessive - potentially leading to excessive vegetative growth.   Seeding occurred earlier in 2005 than in 2003 or 2004 (May 6) in an attempt to maximize the length of the growing season.  The plots were seeded using a push-type precision small plot seeder.  Rows were spaced 0.75 m apart with 0.75 m between plants within the row.  There were two rows in each plot giving a total plot area of 12m2.  There were two replicates arranged in a randomized complete block design.    

Emergence percentages for most lines were around 30%.  As the crop was seeded relatively heavily, most lines had to be hand thinned to prevent overcrowding.  Emergence rates of Reddis and Cruz E were poor despite obviously favorable germination conditions.  This suggests problems with seed quality -   particularly as these lines also had emergence problems in previous years.
   
As milk thistle is slow to establish, some weed control was needed.  However, the wide row and plant spacings made tillage simple. No problems with weed competition occurred in 2005 - although there was obviously competition between milk thistle plants within the row.  No herbicides were applied in this trial in order to avoid confusing agronomic performance with issues of herbicide sensitivity.     
 
Although growers have reported problems with Aster Yellows in Milk thistle, no problems with disease were observed in this trial.  A heavy infestation of Painted Lady Butterfly occurred throughout Saskatchewan in 2005.  Milk thistle is one of the preferred food sources for this occasional pest.  The larvae began to emerge in late June at which time the plants were about 30 cm tall and growing vigorously.  Although the larvae caused significant defoliation - their lifespan is limited and it is doubtful that the feeding damage would have any longterm impact on a plant as vigorous as milk thistle.  Nonetheless - we sprayed the plots with insecticide (Decis) is late June.  A single application provided excellent pest control.  Organic products like Bt. would likely provided adequate control - if problems with Painted Ladies become common.

Note - Milk thistle growers in other areas of the province lost their crop to Painted Lady Butterflies in 2005.     

The plot area was not irrigated in 2005.  This reflects the fact that; a) 2005 was fairly cool and wet and b) results from previous years suggested limited need or benefit of irrigation.  Although there were indications of depletion of soil water by late August, the plots were not irrigated in an effort to promote maturity.   

By late-August some of the lines were approaching maturity.  On Sept 5 the plots were sprayed with the chemical desiccant Reglone (1 l/a) in 100 l/a of water.  Although most lines were still growing vigorously at the time of desiccation - this single Reglone application provided excellent top kill.  All the tops were dead within 3 days of treatment.  The plots were harvested on Sept 8, which is one month earlier than the harvest in 2004.  The entire trial was harvested using a Wintersteiger Nursery Master Elite small plot combine.  The header was raised to minimize the volumes of vegetative material taken in along with the seed heads.  The combine was adjusted to minimize seed losses by reducing the fan speed and opening the sieves.  The harvesting operation was both quick and efficient.  The majority of seed losses reflected shattering prior to the harvest, rather than seed missed by the combine.  Few of the lower canopy heads missed by the header contained any useful seed.  The harvested material was air dried at 40 C for 5d, threshed and then cleaned using a dockage sorter.   Seed yields and 1000 seed weights were determined at this point.  For the quality analysis a seed sample was further cleaned by hand to eliminate all impurities.    

The stand, maturity index and yield data for 2005 again show that some milk thistle lines were far better suited to SK growing conditions than others (Table 1).  Although 2005 was intermediate in temperature between 2003 (hotter) and 2004 (cooler), the 2005 crop was higher yielding with more advanced and uniform maturity than either the 2003 or 2004 crops.  This reflects both the growing season and improvements in production practices.   One line (Genesis seeds) was overmature by the time of the harvest - and losses due to shattering may have been higher than normal in this line (* in table).  As noted in previous trials, some lines were just beginning to flower at the time of the final harvest.     

When seed yields are considered;
a) Milk thistle appears to be capable of producing acceptable yields even with limited plant populations.  Once established, the plants grow rapidly and make efficient use of all available growing space.  Consequently, high seeding rates and/or high rates of seed germination  are not required for good yields.   
b) several of the lines had seed yields that exceeded 1500 kg/ha.  This is promising as the yield levels needed for milk thistle to represent an economically viable crop in Saskatchewan are ca. 500 kg/ha.  It is important to note that yields in small plot field trials are a questionable indicator of the yields that can be expected under commercial production conditions.  However, the fact that overall yields increased significantly over the 3 years of these trials suggests that the utilization of better management practices and greater familiarity with the needs of the crop, could result in even higher yields.    
                        c) there was again little correlation between seed size and yield  

The quality data for the various milk thistle lines tested in 2005 are presented in Table 2, along with the quality data for selected treatments from the agronomy trials.

Some key quality observations from the 2005 germplasm evaluation trials are;
a) The average silymarin content in 2005 was 1.5% compared with 1.4% in 2004 and 1.6% in 2003.  It is interesting to note that the silymarin content appears to reflect the trend in growing season temperatures ... 2003 > 2005 > 2004.   The silymarin content of milk thistle seed is known to increase as the seed matures.  These data support that concept and points out the need to utilize cultivars and production practices that maximize seed maturity within the available cropping season.   
b) relative seed quality of the cultivars was not overly consistent from year to year.  For example, although there were many cultivars that had more than 2% silymarin in a given test year, none of the cultivars exceeded 2% silymarin across all three test years.  This lack of consistency will represent a major challenge if the marketplace focuses on a high silymarin content as an indicator of quality.  At present, the market seems to either not consider silymarin content or is content if the crop exceeds some minimum level ie; 1.6%   
d) total silymarin yield (seed yield * silymarin content) is another potential measurement of a cultivar’s value ... assuming that silymarin content is a factor in sale price.   In the 2004 trial, Austra Hort, Bolier and Kalyx-1 were outstanding for total silymarin yield.  In the 2005 trial,  Austra Hort and Kalyx-1 were again amongst the top ranking lines for total silymarin yield.  Bolier had only mediocre yields and seed quality in 2005.  The highest yielding line in 2005 (Royston Petrie 131) was not tested in 2004 ... but had performed well in 2003.

           
Table 1.  Growth and yield characteristics for Milk Thistle lines in 2005.

Table 2.           Quality characteristics for Milk Thistle lines evaluated in 2005

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New Milk Thistle Lines Released by the University of Saskatchewan

The objective of this program was to develop lines of milk thistle (Silybum marianum) that were suited to mechanical harvesting. Short stature plants with uniform seed maturity and good seed retention within the seed head are all desirable characteristics in a mechanically harvested crop.   Early maturity, high seed yields with acceptable seed quality were also selection criteria.  

Line                Characteristics

U of S-1          Strengths -       High yields.  Short stature and early, uniform maturity.  Good total silymarin content and silybinin B levels are above average.
                        Limitations -    Relatively light seed coat color.

U of S-2          Strengths -       Exceptionally high levels of silybinin B and overall silymarin content.  Matures early.  Represents the only U of S line being released                                                  with seed coat darker than industry standard (Richters).
                        Limitations -    Low rates of seed emergence may limit yields.

U of S-3          Strengths -       Above average yields, medium stature, uniform early maturity.  Slightly above average silybinin B levels.
                        Limitations -   Light seed coat color.

U of S-4          Strengths -       Good yields. Above average silybinin B.  Fairly dark seed coat. 
                        Limitations -    Larger, later maturing plant, but still earlier and shorter than the  industry standard (Richter's).

Table 1.          Characteristics of new U of S Milk Thistle Lines.  Richters line of milk thistle is included as a comparative standard.         

All data are averaged over 2 or more crop years.
Maturity = % of heads reaching maturity by the 2nd week of Sept.

All lines have been licensed to the Saskatchewan Herb and Spice Association (SHSA)
Contact : Connie Kehler - Manager of the SHSA for information on seed suppliers 

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Milk Thistle Agronomy Trials (2003-2005)

Jazeem Wahab (CSIDC, Outlook, SK)
Doug Waterer (University of Saskatchewan, Saskatoon, SK)

Background

Milk thistle is native to the Mediterranean, but is now widespread throughout the world. This stout thistle usually grows in dry, sunny areas. The stem branches at the top and reaches a height of about 1.5 to 4 m. The leaves are spiny and wide, with white blotches or veins. The flowers are red-purple. The small, hard-skinned seed is brown, spotted, and shiny.  Milk thistle is an easy to grow annual plant.  It has an indeterminate growth and flowering habit, resulting in uneven development and maturity of flower heads. 

A group of flavenoids called “silymarins” represent the medically valuable compounds sought after in milk thistle.  Silymarins have been demonstrated to both protect and alleviate problems with liver and kidney function.  Silymarins are present throughout the milk thistle plant, but occur at the highest concentration in mature seed.  By comparison, immature seed contains relatively little silymarin.  Consequently, the timing of seed harvest is critical.  However optimum harvest timing is complicated by the fact that;
            a)  milk thistle is a late maturing plant with an indeterminate flowering habit.
            b) milk thistle is prone to shattering once the seeds are mature

At present, milk thistle is harvested by hand-cutting each seed head as it matures.  This process is slow and costly, particularly as the plants sport very large and sharp thorns.  Saskatchewan growers are struggling to effectively compete with other potential suppliers of milk thistle who have access to more available and affordable sources of hand labor. 

Once-over mechanical harvest of milk thistle is possible but yields can be less than 50% of those achieved by hand harvesting (Wahab 2002).  The quality of the resulting crop is also poor as the once over harvest combines mature and immature seeds.  The practical and economic viability of mechanical harvesting milk thistle may be improved if ...;

a) lines of milk thistle with more uniform flowering habit are identified.   This objective was addressed in the germplasm evaluation component of this report.

b) agronomic practices are identified that enhance uniformity of crop development and flower maturity  

c) pre-harvest desiccation is used to accelerate and synchronize maturity of the flower heads.   

 The objectives of this project include:

i.    Seeding rate and row spacing effects on yield and quality.
ii.   Seeding date effects on growth characteristics and productivity.
iii.  Nitrogen and phosphorus effects on productivity.
iv.  Effectiveness of vinegar as a desiccant for machine harvest compared to Reglone.

Study Description

Agronomic studies were conducted 2003 through 2005 at the Canada-Saskatchewan Irrigation Diversification Center in Outlook Saskatchewan.  All studies were conducted under dryland conditions, as previous experience at CSIDC showed that irrigation can prolong vegetative growth and delay maturity of milk thistle, resulting in considerable yield losses.   The soil at the test site is a clay loam.  The soil properties at the test locations are summarized in Table 1.  Land was prepared in the traditional manner to form a firm seed bed.  Milk thistle seed from Richter’s Herb Co. was used in the studies.  This line was selected as it has produced reasonably good yields in previous trials and has a good quality profile.  Each test plot was 3.7 m x 1.2 m.  Seeding was done with a double-disc press drill and seeds were placed approximately 1-2 cm deep.  Rows were spaced 60 cm apart with seeding rate of 100 seeds/m2 for all studies except for the seeding rate and row spacing study. 

Milk thistle initially grows relatively slow but once established it grows vigorously and competed very well against late season weed pressure.   One early weeding was sufficient.   The crop was desiccated when the flower heads matured, i.e. formation of pappus on the seed.  During maturity, the purple petals dried and white pappus appeared; the seed turned from light brown and soft texture to dark brown and hard. 

Two desiccants, Reglone (2.7 l/ha in 1000 l water/ha) and vinegar (14% acetic acid at 1000 l/ha) were sprayed for desiccation during the appropriate stages based on the trial, i.e  when flower heads were 30% and 60% mature.  Seeds were harvested using a Wintersteiger plot combine.  

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2003 Studies
           
I.         Effects of nitrogen, phosphorus and harvest stage on yield and quality of milk thistle:

Three levels of pre-plant nitrogen (0, 50, 100 kg N/ha.) and three levels of phosphorus (0, 60, 120 kg P2O5/ha) were examined in this study.  Field plots were laid out as a 3 (nitrogen) x 3 (phosphorus) factorial in a Randomized Complete Block Design with four replications.  Two similar trials were conducted for desiccation at two different stages (30% and 60% mature flower heads) using Reglone.  Seeding was done on May 23, 2003.  The first desiccation (‘Early’) was carried out when approximately 50%-60 % of the flower heads were mature (September 2, 2003) and the second desiccation (‘Late’) six days later (September 8, 2003).  The ‘Early” desiccated crop was harvested on September 5, while the ‘Late’ desiccated crop was harvested September 12.  

II.        Seeding rate and row spacing effects on yield for milk thistle desiccated with vinegar. 

Six seeding rates (25, 50, 75,100, 125, 150 seeds / m2) and two row spacing (20, 60 cm) were evaluated.  Field plots were laid out as a 6 x 2 factorial in a Randomized Complete Block Design with four replications.  Seeding was done on May 23,  2003.  The crop was desiccated with Vinegar on September 12, 2003 (when 50%-60% of heads were mature) and harvested on September 19, 2003.

2004 Studies:

I.         Effects of nitrogen, phosphorus on yield and quality of milk thistle when desiccated with vinegar and Reglone at two maturity stages:

Three levels of pre-plant nitrogen (0, 50, 100 kg N/ha.) and three levels of phosphorus (0, 60, 120 kg P2O5/ha) were examined in this study.  Field plots were laid out as a 3 (nitrogen rate) x 3 (phosphorus rate) factorial in a Randomized Complete Block Design with four replications.  Four similar trials were conducted for the combination of the two desiccants (vinegar and Reglone) and two stages of crop maturity (30% and 60% maturity).  Seeding was done on May 14, 2004.  The ‘Early’ desiccation (30% maturity) was carried out on September 7, 2004 for Reglone, and on September 8, 2004 for vinegar.  The ‘Late’ desiccation (60% maturity) was carried out on September 15, 2004 for both vinegar and Reglone.  Seed harvest was taken on September 27, 2004. 

II.        Seeding rate and row spacing effects on yield for milk thistle desiccated with vinegar. 

Six seeding rates (25, 50, 75,100, 125, 150 seeds / m2) and two row spacings (20, 60 cm) were evaluated.  Field plots were laid out as a 6 x 2 factorial in a Randomized Complete Block Design with four replications.  Seeding was done on May 14, 2004.  The crop was desiccated with Vinegar on September 15, 2004 (when 50%-60% of heads were mature) and harvested on September 24, 2004.

2005 Studies:

Studies conducted during the summer of 2005 were similar to those conducted in 2004.  Test plots were seeded on May 13, 2005.  The ‘Early’ desiccation (30% maturity) was performed on August 27, 2005 and the ‘Late’ desiccation (60% maturity) was performed on September 1, 2005.  The seed crop was harvested on September 19 and 20, 2005.
                                   
Fall Seeding Study:

Studies were also conducted in 2003 and 2004 to examine the feasibility of seeding milk thistle during the fall.  The objective of fall seeding was to advancing flowering and consequently harvest period.  Identical seeding rate and row spacing combinations similar to spring seeding were used for the fall seeding studies. 

Cropping conditions at the site were generally suited to milk thistle production.  Crop health was good is all years.  The 2003 growing season was relatively warmer and dryer than 2004 or 2005 (Figures 2 and 3).  The 2003 growing season received 133 mm of rain compared to 304 mm in 2004 and 391 mm in 2005.  

On average, the 2004 season produced higher seed yields relative to 2003 or 2005.  The 2005 crop had the potential for excellent yields but a heavy rain and wind storm on September 11  (69.2 mm) caused substantial seed loss from the mature heads.  Yields in 2005 were consequently poor. 

Figure 2.   In-season rainfall during the 2003, 2004, and 2005 growing seasons.

Figure 3 Average maximum temperature (a) and minimum temperature (b) during the growing seasons: 2003, 2004, and 2005.

Seeding Rate and Row Spacing Effects:

Studies conducted 2003 through 2005 examined seeding rates ranging from 25 to 150 seeds per m2 and 20 cm and 60 cm row spacings.  Seeding rates and row spacing had no effect on seed yield during the three years (Table 2).  Between the seeding rates tested, seed yields ranged between 602 kg/ha and 710 kg/ha in 2003, 983 kg/ha and 1015 kg/ha in 2004, and 318 kg/ha and 387 kg/ha in 2005. 

The two row spacings tested, i.e. 20 cm and 60 cm produced similar seed yields during the three years (Table 2).  Although the effect was non-significant, the 60 cm row spacing produced higher seed yield than the 20 cm spacing in 2003, while the 20 cm spacing produced slightly higher yields than the 60 cm spacing during 2004 and 2005.

Nitrogen and Phosphorus Rate Effects:

The yield response to rates of nitrogen and phosphorus application in combination with the type of desiccant (vinegar, Reglone) and desiccation timing (30% and 60% maturity of flower head) during the 2003, 2004, and 2005 growing seasons are summarized in Tables 3, 4 and 5 respectively. 

During 2003, application of 100 kg N/ha produced the lowest seed yields compared to 50 kg N/ha or no nitrogen control (Table 2.3).  In 2004 and 2005, nitrogen application in a few instances tended to produce slightly higher yield than the no nitrogen control (Tables 4 and 5).

Phosphorus application rate had no effect on seed yield of milk thistle across the three years (Tables 3, 4, and 5).

Comparison of Vinegar and Reglone as Desiccants and Timing of Desiccation:

Application of Reglone at 2.7l/ha @ 1000 l water/ha was a more effective desiccant than the commercial grade vinegar, i.e.14% acetic acid  (Figure 4). 

Despite the large plant size and crop vigor at the time of treatment, a single application of Reglone usually produced adequate dry down.  The fact that relatively large volumes of water were used likely enhanced the efficacy of the Reglone treatment.  It was possible to combine the Reglone treated crop within 3-7 days after spraying depending the weather conditions.  Dry down was faster when the weather was warmer compared to cooler weather conditions. By contrast, vinegar was not an effective means of top-kill of milk thistle .  Even after two applications, the dry-down was insufficient after two weeks for proper machine harvesting. 

On average, desiccation with Reglone produced 23% higher seed yield in 2003 and 22% higher seed yield in 2004 than using the vinegar.  This reflected seed loss during the extended dry down period required for the vinegar treatment. However in 2005, desiccation with vinegar resulted in 15% higher seed yield than desiccation with Reglone.  This is likely due greater shattering loss in the drier Reglone desiccated crop during the rain event that occurred just prior to harvest in 2005. 

Delaying desiccation, from 30% mature heads to 60% mature heads, produced higher seed yields in 2003 and 2004 for both methods of desiccation.  In 2005, the early treatment increased yields in the Reglone desiccated plots - this again likely reflects differences in shattering loss during the fall storm event (Tables 3, 4 and 5).

Fall Seeding:

Fall seeding was attempted for milk thistle during two years with the objective of achieving early crop establishment - thereby potentially increasing yields and seed quality.  Germination and stand establishment were extremely poor in the fall seeded crop in comparison to spring seeding (Figure 5).

Summary:

- Milk thistle shows little yield response to a wide range of in and between row seed spacings.  As seed is large and costly, low seeding rates (25 seed/m2) appear sufficient.
-  20 and 60 cm row spacings produced similar seed yields.  The wider row spacing allowed more room for early weed competition, but it also facilitated mechanical tillage to remove the weeds.
-  Fall seeding was not successful, as stand establishment was extremely poor compared to spring seeding.  The fact that volunteer milk thistles are uncommon despite the fact that the crop  shatters extensively also suggests limited overwinter survival of the seed.  
- Milk thistle shows little yield response to nitrogen or phosphorus fertilizer application on a site with fairly high background levels of soil fertility.   Fertilizer applications did tend to increase vegetative growth of the crop - but this was often detrimental as it made harvesting more difficult.  Fertility levels had no impact on levels of bio-active compounds in the seed.
- The crop has to be desiccated prior to machine harvest.
- Delaying desiccation until 60% of the flowers had begun to dry down produced higher seed yields and superior seed quality (higher silymarin content).
-  Harvest timing is critical.  Inclement weather when the crop is ready for harvest (rain, wind etc.) can cause severe yield losses due to seed shattering.
-  Reglone is an efficient and cost-effective desiccant for milk thistle. 
-  Commercial grade vinegar (14% acetic acid) can be used as an organic desiccant, but it is expensive, slow acting and results in increased seed loss to shattering as compared to desiccation with Reglone. 

Conclusions - Over the 3 years of this study we examined the seed yield potential and level of active ingredient (% silymarin) in a range of selections obtained from commercial suppliers, breeders and gene banks.   There was very significant variability in the performance of the material available.  Some the lines failed to mature within the growing season available in Saskatchewan, while others were fully mature by late August.  There was also significant variation in seed quality as measured by the silymarin content.  There was no real association between yield potential and seed quality.   The relative performance of the lines tested was not overly consistent from year to year in terms of either their yield potential or seed quality.  This made it difficult to select for clearly outstanding lines.  In discussions with members of the Saskatchewan Herb and Spice Growers who are marketing Milk Thistle ... there are at present no standards being stipulated for minimum silymarin content.  In that situation, lines with exceptional yield potential (ie; Austra Hort, Bolier or TMP 14068) would be best.  In situations where quality is also considered in developing a market price, lines that have both yield and quality would be required.  Austra Hort and  Kalyx-1 have the desired combination of yield potential with seed quality. 

Wanda Wolf (pers comm 2002) estimated the break-even yield for hand harvested milk thistle at 500 kg/ha.  Yields of some of the better adapted lines in this trial were around 1500 kg/ha.  At a market price of $ 8.00 CDN/lb, a 1500 kg/ha milk thistle crop has a theoretical net return potential of $14,200/ha.

Seeding and stand establishment are straight forward for milk thistle and the crop has no problems out-competing weeds.  Fertility responses were minimal or negative (reduced stand and delayed maturity) .  Yields were not affected by either in row or between row spacings.   Milk thistle appears to be a very plastic crop, adapting to the growing space available.  As the seed is large and costly, the data suggests that a low seeding rate and wide row spacing would be most economical without compromising yield potential of the crop.  There were no significant disease problems observed and any insect pests were easily managed.

Increasing the duration of the growing season, either by early planting or delayed harvest increased yields and the concentration of active ingredients in the seed.  However, shattering of over-mature seed heads appears to be the greatest production threat for milk thistle.  The highly desirable early maturing, large seeded lines appeared to be particularly prone to shattering.  Timing and timeliness of  harvest is critical - to minimize the risk of shattering, the crop should be harvested within a few days of the seed pods reaching maturity (60% fluff) - either natural maturity or maturity triggered by frost or application of a chemical desiccant.  Once the crop reaches that stage of maturity, any delay in harvesting can result in substantial seed loss.  The desiccant Reglone provides a very quick die down of milk thistle - and consequently shattering losses are minimal.  However, Reglone is not approved for organic production.  Although application of high rates of vinegar did eventually result in desiccation of the crop, shattering losses during the extended dry down period were excessive.

Milk Thistle is traditionally hand harvested - with each seed head individually harvested as it reaches maturity.  Although this may increase yields and enhance uniformity of seed maturity, it is not a practical option in Saskatchewan.  Not only would hand harvesting be prohibitively expensive, it is doubtful that a labor pool could be identified that would be willing to work with the extremely sharp thorns on the seed heads.  Mechanical harvest using standard small plot combines appears to be a viable alternative - providing that the crop has been desiccated (by frost or chemically) prior to combining.  In two years of trials, a once over harvest with a standard small plot combine produced yields that were comparable or slightly higher than a selective hand harvest. Obtaining a yield advantage by combining was unexpected.  While harvesting the individual mature seed heads, the hand harvesters may have been bumping adjacent heads causing them to shatter out. Several of the highest yielding lines identified in this project also appeared well suited to mechanical harvesting - in that they matured uniformly, were not excessively vegetative and did not shatter prior to harvest.   Milk thistle was easily threshed, providing the tops were dead prior to harvest.   Cleaning and sorting of the seed is readily accomplished using standard equipment.  Some upgrading of quality is possible by seed sizing - but color sorting does not look to be a straight forward option.    

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Seeding Rate and Desiccation Method Trials for Milk Thistle (2007 and 2008)

Jazeem Wahab (CSIDC, Outlook, SK)
Doug Waterer (University of Saskatchewan, Saskatoon, SK)

Summary
Field trials conducted in 2007 and 2008 sought to develop appropriate agronomic management practices for the new improved lines of milk thistle recently generated by the University of Saskatchewan.  Seed yields from the new shorter stature lines were generally superior to the standard cultivar (Richters) and the seed also contained higher concentrations of the target phytochemicals.   Seed yields and seed quality increased with increasing plant populations to a peak around 100 seeds planted/m2.  This yield and quality response may reflect the more uniform flowering and seed development achieved when branching of the milk thistle plants is suppressed by growing in a dense stand.  Pre-harvest desiccation was essential to mechanical harvest of the milk thistle crop.   Diquat (reglone) and concentrated acetic acid both provided an acceptable degree of desiccation.  Diquat is faster acting and less expensive than acetic acid, but acetic acid is perceived more favorably in organic markets.     

Background
There is substantial worldwide demand for milk thistle (Silybum marianum) seed as a herbal medicine. Both ‘conventionally’ and ‘organically’ grown milk thistle are sought by the market place.  The University of Saskatchewan has recently identified superior quality high yielding locally adapted germplasm suited for mechanical harvest under Saskatchewan growing conditions.   The Saskatchewan Herb and Spice Association has been granted exclusive rights to grow these new lines. 
Previous research conducted at the Canada-Saskatchewan irrigation Diversification Centre demonstrated that there is excellent potential for mechanized production of high quality milk thistle in Saskatchewan even using the relatively unsuitable cultivars previously available (c/o Richters seed).  One of the main objectives of the present project is to develop agronomic refinements needed to maximize yields and quality of the new milk thistle lines selected at the University of Saskatchewan.  

Saskatchewan’s relatively short growing season combined with uneven maturity of milk thistle is a major challenge for production, particularly if the objective is to harvest the crop via once-over combining.  By increasing the plant population in the field it may be possible to reduce branching, thereby better synchronizing flowering, resulting in more  uniform crop maturity.    More uniform maturity should facilitate machine harvest while also reducing shattering loss and improving the overall quality of the harvested seed.      

Due to its robust growth habit and indeterminent flowering pattern the milk thistle crop has to be desiccated prior to harvest.  Previous research conducted at the CSIDC and University of Saskatchewan has demonstrated that Reglone (diquat) is an effective desiccant for milk thistle.  Reglone is presently registered under regulatory review for use in Canada as a milk thistle desiccant.   However, the market preference for organic milk thistle necessitates the identification of organic desiccants for top-kill. 

This project investigated agronomic practices designed to further increase yields and quality of selected lines of milk thistle in Saskatchewan, and also examined the effectiveness of vinegar as an organic desiccant.     

All the milk thistle lines utilized in this project are under exclusive licence to the Saskatchewan Herb and Spice Association - therefore, all line specific information generated in this project will be of direct and exclusive benefit to Saskachewan growers of this crop.  

Methods  

Field trials were conducted during the summers of 2007 and 2008 at the Canada-Saskatchewan Irrigation Diversification Centre, Outlook.   Six milk thistle cultivars, (U of S-1, U of S-2, U of S-3, U of S-4, Lone Wolf, and Richters) were evaluated in 2007.  Due to the poor performance of the Lone Wolf selection in 2007, this selection was omitted from the 2008 trials.  Five seeding rates (25, 50, 100, 125, and 150 seeds/m2), two desiccants (Reglone (conventional) and Vinegar (organic) were evaluated for each cultivar separately.  Field plots were laid out on Split-plot Design with four replications.  Main-plot consisted of Desiccants and the Sub-plot consisted of seeding rates.  Individual plots (Sub-plots) were 3.7 m x 1.2 m.

The soil at the test site during the three years was a clay loam.  In the spring the land was prepared in the traditional manner to form a firm seed bed.  Plots were seeded using a double-disc press drill with seeds placed at approximately 1-2 cm depth.  Rows were spaced 60 cm apart with appropriate seeding rates as defined by the treatments.  The crop was raised under dryland growing conditions, as previous experience at CSIDC showed that irrigation of milk thistle prolonged vegetative growth and delayed maturity, resulting in considerable yield loss.  The only exception was that two light irrigations were applied after seeding to ensure germination and proper crop establishment.  One weeding during early growth stage was sufficient to raise the crop.  The crop was desiccated using Reglone and Vinegar when approximately 50% of the flower heads had matured i.e. formed of pappus on the fruit. The application rate consisted of 2.7 l/ha of Reglone applied in 1000 l water/ha during 2007 and 2008, and vinegar, (14% acetic acid at 1000 l/ha in 2007, and 60% acetic acid at 1000 l/ha in 2008).  The crop was harvested once the stalks were sufficiently dry to pass through a Wintersteiger plot combine.

Silymarin content of the harvested seed was analysed by Phytovox Inc., Edmonton, Alberta.  Pooled samples from all replicates of the lowest (25 seeds/m2) and highest (150 seeds/m2 ) seeding rates, the two desiccation treatments (Reglone and Vinegar) for each of the different milk thistle lines were analysed for Taxifolin, Silychristin, Silybin A, and Silybin B.

Trial Details

2007. 
The test plots were seeded on May 18, 2007 using a Fabro seeder.  Two light irrigations were applied after seeding to ensure germination and proper crop establishment.  The crop was otherwise grown under dryland conditions, based on previous experience with milk thistle.

The 2007 growing season was relatively warm with July being exceptionally warm and dry.  The crop received 229 mm of rain (Figure 1).  Hail storms on July 31, 2007 and August 18, 2007 caused considerable damage to flower heads, negatively affected seed filling and resulting in substantial yield losses.  The crop was desiccated on August 14 with Reglone and vinegar at appropriate concentrations.   Seed was harvested on August 30, 2007 and 31, 2007 using a Wintersteiger plot combine.  The harvested seed was dried at 35 degrees C.  The seed was cleaned and seed yield was recorded.

2008.
Pre-plant soil analysis at the test site indicated 71 kg NO3-N/ha, 59 kg P/ha, 370 kg K/ha, and >86 SO4-S/ha at 0-30 cm depth.  No additional fertility was provided.  The test plots were seeded on May 14, 2008.  Two light irrigations were given soon after planting to ensure germination and proper crop establishment.  Subsequently, the crop was grown under dryland condition.  Flowering occurred around July 21 to July 28 and the crop was ready for desiccation three to four weeks later.  The crop was desiccated on August 22 & 23. 

NB – whereas in previous trials 12-14% non-synthetic or “natural” acetic acid was used as the organic desiccant, in 2008  60% (synthetic) acetic acid was used.  The concentrated acetic acid was applied at the same rate as used in 2007 (1000 l/ha).  It should be noted that the synthetic acetic acid is not on the list of materials approved for use within “organic production systems.  

Both desiccation treatments were ready for combining within a week of treatment (August 29, 2008).

The growing season was relatively warm and received 131 mm of rain during the crop growth period.  Hail storm on August 21, 2008 caused slight damage to the crop.

Results and Discussion

Growing Season Climatic Conditions:
The 2007 and 2008 growing season temperatures were relatively similar, with July being fairly warm (Figure 1).  The crop received 229 mm of rain in 2007 and 137 mm in 2008 (Figure 2).

In 2007, hail storms on July 31 and August 18 caused considerable damage to flower heads that negatively affected seed filling resulting in substantial yield losses, but not complete crop failure.  No hail was experienced in 2008. 

Seed Yield:
In 2007, the two hail events caused early death of the crop and also caused significant amounts of shattering of seed from the seed heads.  In 2008, despite lower rainfall seed yields were substantially higher than 2007.  This confirms the fact that inclement weather during the latter stages of the crop, particularly after maturity, can cause considerable crop losses.

The effects of desiccation methods and seeding rate on seed yield during 2007 and 2008 for the various milk thistle selections are summarized in Table 1 and Table 2 respectively.  The corresponding effects of treatments on average seed weight are summarized in Table 3 and Table 4 respectively. 

Cultivar Response:
In 2007, the selection U of S-2 produced the highest average seed yield of 200 kg/ha, while the cultivar from Lone Wolf produced the lowest yield of 13.4 kg/ha (Table 1).  The yield ranking was U of S-2 > U of S-4 > U of S-1 > Richters > U of S-3 > Lone Wolf. 

In 2008,  the average seed yield for the different selections ranged from as low as 756 kg/ha for U of S-1 up to 946 kg/ha for U of S-4 (Table 2).  The yield ranking was U of S-4 > U of S-3 > Richters > U of S-2 > U of S-1.  These seed yields are comparable to yields obtained in previous “successful” years of trials in Saskatchewan.   

Seeding Rate Effects:
Seeding rate significantly affected seed yield in both 2007 and 2008 (Tables 1 and 2).  Yield responses to seeding rate were variable for the different milk thistle selections during the two years.  The mathematical relationships between seed yield and seeding rate during 2007 and 2008 for U of S-1, U of S-2, U of S-3, U of S-4, and Richters are presented in Figs 3-7 respectively.  The responses were variable for U of S-1, U of S-2, and U of S-3 between the two years. For example, the seed yield for these selections increased in a linear manner with increasing seeding rate in 2007 and assumed a quadratic relationship in 2008.  For U of S-4 and Richters, the seeding rate response was quadratic in both years.  However, it is not clear why the quadratic responses were so dissimilar over the two years for the Richters line of milk thistle (Figure 7).

The optimum seeding rate for the various cultivars can be estimated from the vertex of the yield response curve (Table 3).  The optimum seeding rate for the various milk thistle lines was somewhat variable but it appears that a seeding rate between 100 to 125 seeds per m2 tended to produce the highest seed yields

The two desiccation methods used, i.e. Reglone or Vinegar, provided an equivalent degree of crop desiccation and had no effect on seed yield in 2007 and 2008 (Tables 1 and 2).  In 2007, the crop had been severely damaged by hail prior to application of either top-killing treatment.  This hail damage may have rendered the crop susceptible to even the relatively mild effects of desiccating with relatively dilute (12%) acetic acid.

In previous trials (see Milk Thistle Agronomy) the Reglone treatment had consistently produced higher seed yields than the organic desiccant.  This yield advantage was attributed to the much more rapid desiccation achieved with Reglone – as a rapid drydown would reduce the time during which the mature seed could shatter out.  In the 2008 trial the drydown achieved with the “organic” desiccant was as quick and thorough as that achieved with the Reglone.  This may be atrributed to (i) the change over to more adapted, earlier maturing milk thistle lines, (ii) use of production practices conducive to acclerated crop maturity (ie; reduced fertility, minimal irrigation, high seeding rates) and c) the use of much more concentrated acetic acid (60%) in 2008 versus 12% acetic acid in 2007.   

Average Seed Weight:
Seeding rates and desiccation methods had no effect on seed weight for any of the milk thistle lines tested in 2007 and 2008.  Average seed weight (g/1000 seed) for the different selections varied between 16.5 to 18.4 g in 2007 (Table 4) and from 14.7 to 22.2 g in 2008 (Table 5).  U of S 3 produced the smallest seed (14.5 g/1000 seed); U of S 2, U of S 4, and Richters produced larger seeds (20.5 - 22.2 g/1000 seed), and U of S 1 produced intermediate seed (16.5 g/1000 seed).

Quality (Flavonoids):
The flavonoid profile for the various milk thistle selections was influenced by seeding rates and desiccation methods.  These results are summarized in Table 6 (individual values) and Table 7 (treatment averages). 

This preliminary analysis showed the following trends with respect to the effects of treatments on flavonoid for the various milk thistle selections:

-  seeds from U of S 2 at 150 seeds/m2 desiccated with Reglone and U of S 1 at 25 seeds/m2 also desiccated with Reglone contained the highest and lowest amounts of all the flavonoids respectively.  The total flavonoid levels ranged between 11.48 and 21.66 µg/g (i.e. 1.2% and 2.2%) of seed dry weight. 

- U of S 2 contained the highest amounts of Taxifolin, Silychristin, Silybin A, Silybin B, and Total flavonoids relative to the other selections.  In previous trials U of S 2 had consistently produced superior levels of the various bioactive molecules (see Milk Thistle Agronomy). 

- seeds harvested from plots with the highest plant population (150 seeds/m2) had higher flavonoid levels than seed from plots with the lowest plant population (25 seeds/m2).   This may reflect the earlier and more uniform seed maturity achieved with a higher plant population.   The flavonoid content of milk thistle seed is know to increase with seed maturity, although it peaks and stabilizes well before the seed is ready to drop from the seed head.   The relative amounts of the different flavonoids are also known to change with seed maturity, however these changes are also strongly influenced by genotype, growing conditions and method of extraction and analysis.    There is still some considerable debate within the industry as to which of the flavonoid components is the best indicator of “potency” of the milk thistle extracts.  Until this debate is settled, the overall silymarin content is considered to be the “default” indicator of quality. 
 
- there was no difference in flavonoid levels between the two desiccation treatment (Reglone or Vinegar).

 
Conclusions

The objective of the agronomy trials conducted in this project was to develop appropriate management practices for the improved lines of milk thistle recently generated by the University of Saskatchewan.  The new milk thistle lines produced a vigorous stand of relatively short stature evenly maturing plants that were better suited to mechanical harvest than the line previously used as the industry standard (c/o Richters).  Seed yields from the new lines were generally superior to the standard cultivar, although the performance of all lines varied from year to year.  The nature of this genoype X environment  interaction needs to be further explorer in order to increase confidence in the performance of a specific line(s).   The seed harvested from the new lines also contained higher concentrations of the target phytochemicals than the standard.    The combination of improved yields and superior seed quality indicates that the new lines created by the University of Saskatchewan are meeting expectations.   In particular, the line UofS-2 has emerged as providing a combination of superior seed yields and seed quality in a vigorous but readily managed short stature, fast maturing plant phenotype. 

Seed yields and seed quality increased with increasing plant populations to a peak at around 100 seed/m2.   This yield and quality response to plant population may reflect the more uniform flowering and seed development achieved when branching of the milk thistle plants is suppressed by growing in a dense stand.    The dense stand also reduced weed competition early in crop development.  While the heavy seeding rate would increase seeding costs, the corresponding yield and quality advantage is clearly sufficient to offset this cost.

This study, as well as previous research showed that there was a significant year X genotype interaction for both the total concentration and the relative amount of different bioactive flavonoids found in the milk thistle seed extract.   It also appeared that the flavonoid content and composition could be influenced by plant populations but was not influenced by the method of desiccation.   These interactions complicate the process of identifying and adopting genotypes and production practices that optimize product quality.  This process is further complicated by the fact that the industry has not been able to identify which of the flavonoid components is the best indicator of “potency” of the milk thistle extracts.  Until this debate is settled, the overall silymarin content should likely be considered to be the “default” indicator of quality.   This project has identified genotypes and production practices designed to maximize both yields and product quality.     

While the new short stature, even maturing milk thistle lines are clearly better suited to mechanical harvesting than the previous standard lines, pre-harvest desiccation is still essential to efficient mechanical harvest of the milk thistle crop.   Diquat (reglone) and concentrated acetic acid both provided an acceptable degree of desiccation.   It should be noted that thorough coverage is essential for effective kill down of a vigorous milk thistle crop – and this coverage hinges on the application of very large volumes of the desiccant (1000L/a).   Growers must be cautioned against trying to scrimp on the desiccant volume – otherwise the top-kill process will be compromised.  Of the two desiccants tested, Diquat is more readily available, faster acting and much affordable.   Registration of reglone for use in milk thistle is undergoing regulatory review in Canada.   The main reason that a grower would consider use of acetic acid is demand in the organic marketplace.    This raises a potential problem.   In previous research the naturally sourced 12% acetic acid provided a slow and incomplete degree of top kill even at very high rates of application.   While this product provided an acceptable degree of top kill in the 2007 crop it should be noted that this crop had been weakened by a severe hail event and was therefore relatively easy to kill down.   For this reason it was decided to test the efficacy of a much more concentrated form (60%) of acetic acid in 2008.   This formulation provided the very fast and thorough desiccation required for consistent machine harvest of milk thistle.  However, it should be noted that this form of acetic acid is derived from a synthetic process that uses petrochemicals as starting materials.   This effectively disqualifies this product from approval for use in “certified organic” production.    In discussions with the SHSA, the issue of which product a milk thistle crop is desiccated with has not raised any particular concern.  The market is not presently requiring organic certification and is not paying a premium for organically grown milk thistle.   This would suggest that in the present market situation desiccation with either Reglone or concentrated acetic acid are viable options. 

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Milk Thistle Value Added

Value-added Products From Milk Thistle “Waste”

At present, only a small component of the milk thistle plant (silymarin from the seed) has market value.  The seed contains significant qualities of oil - yet this oil is presently regarded as a waste product.  The plant also produces a huge amount of biomass - another waste product unless alternate uses are discovered.  Potential uses of the waste biomass are animal fodder or alternatively a starting point for biofuel - especially as the stems and leaves are rich in high energy latex compounds.  These latex compounds may also have value as a feedstock in the manufacture of industrial materials … like rubber and latex.  At present, there are no crops being grown for latex in Canada - but there is demand for latex as the starting point in the manufacture of rubber and other materials.  The potential to extract products of potential use as fuel, food, fodder and medicine from a single easy to grow plant would appear to make milk thistle an ideal model for of a future bio-based economy. 

This project sought to identify potential value in the “waste products” generated by the milk thistle crop.   

1.0       Recovery of Value-added Products from Milk Thistle

1.1.      Seed oil

The research literature indicates that milk thistle seed contains 12-26% fixed oils, depending on the genotype and the growing conditions.  At present, these oils represent a waste product which is discarded following silymarin extraction from the seed. 

This project evaluated the oil concentration, composition, total yield and potential value of the oils recovered from the newly developed milk thistle lines being commercialized by the SHSA.

Experimental design
            Seed source - SHSA lines generated in agronomy trials
                        Variables examined
                                    - Total oil content
                                    - Oil composition
- Impact of agronomic variables and site/growing season on oil yields

Milk Thistle seed samples covering the previously outlined range of variables (cultivar, site of production, year of production) were sent to Dr. Martin Reaney of the Dept. of Applied Microbiology and Food at the University of Saskatchewan for analysis. 

Oil was extracted from small samples of milk thistle seed using a Goldfisch extraction apparatus and the oil composition was than tested via NMR.   A cold press was used to extract the oil from larger (1 kg) samples of a limited number of seed lots.

Averaged over the cultivars tested, seed oil content was lower in 2004 (20.2%), than in 2005 (25.2%), or 2006 (24.9%)(Table 1.1).  The low oil content in 2004 likely reflects the immature stage of the crop when hit by an early killing frost.  The 2005 and 2006 growing seasons were much more favorable, leading to higher yields of more mature seed with a higher seed oil content.  Differences in seed coil content between cultivars were not consistent from year to year.  Oil yields from cold press extraction were comparable to the yields obtained using the Goldfisch apparatus (Table 1.1).         

Table 2.1.   Seed oil content for various milk thistle lines in 2004-2006. 

NMR analysis of the oils showed no significant differences among the samples (data not shown). The fatty acid profile of all samples was essentially similar (Table 1.2). 

Table 1.2  Fatty acid profile of the oil extracted from the seed harvested from several cultivars of milk thistle in 2004-2006.  

Fig 1.1.  NMR analysis of the oil profile typical of Milk Thistle.

The seed oil content and fatty acid profile of milk thistle oil, along with several of the most commercially important oils used in food production are summarized in Table 1.3.  

Table 1.3.        Seed oil content (%) and fatty acid profile (%) of oil extracted from Milk Thistle seed and several other commercially important oil crops.

The fatty acid profile of the oil extracted from milk thistle was comparable to the oil extracted from corn, sunflower and soybean.  This suggests that the oil would likely have acceptable performance characteristics for use in food processing – assuming that it does not have any unusual/undesirable flavour characteristics.  However, the fact that the oil composition is comparable to mainstream commercial products like corn and soybean oil means that it will be in direct market competition with these low cost alternatives. 

Based on the seed yields and seed oil content seen in this study, oil production/unit area for milk thistle is well below that of the key oil producing crops (Table 1.4).  The low oil yield/unit area, coupled with the fact the fatty acid profile is not unique suggests that growing milk thistle as an oil crop is not an attractive option.  However the results do suggest the potential to use the milk thistle oil that is left over from silymarin extraction as another profit stream – much like the wine industry is capturing value (grape seed oil) from the residues left from their main processing objective.   

Table 1.4.        Oil production/unit area for milk thistle and other oil producing crops 

2.1       Identification/Recovery of other useful by-products from milk thistle

Milk thistle gets its name from the “milky” exudate that oozes from any cuts to the leaves, stems or roots.  The chemical composition of the milk thistle exudate is not known - but closely related species have been identified as sources of latex.  The fact that milk thistle produces a large biomass with minimal management or inputs, suggests its potential efficiency as a ‘bio-factory’.  As the vast majority of the milk thistle plant is, at present, effectively a waste product - any alternate uses for this waste material would represent another potential profit source.
  
Test material grown in 2006 and 2007 by the University of Saskatchewan was made available to Dr. John Balsevitch, G. Bishop and L. Deibert – who are natural products biochemists employed by PBI/NRC, Saskatoon.

Leaves of milk thistle were harvested just past full flowering.  The leaves were allowed to air dry and processed via grinding and followed by extraction with 60% methanol (aq) followed by 100% methanol.  A 50 g sample of dried leaf material yielded after evaporation of the combined methanolic extracts about 15 g of residue. 

The combined methanolic extract was analyzed by HPLC-MS and observed to contain two main flavonoid glycuronides tentatively identified as apigenin and luteolin 7-O-glucuronides (Fig 2.2.1) based on the spectral data and a published report of similar compounds being observed in the flowers.1

In the samples examined the putative “apigenin” was the major flavonoid (ca. 7:3 ratio). 

Processing of the extract via partitioning between ethyl acetate/water and butanol/ water afforded three fractions – ethyl acetate soluble (1.1 g), butanol soluble (1.8 g) and water soluble (12 g).  The ethyl acetate fraction was composed of lipophilic materials including pigments and triterpenes, the butanol fraction was composed largely of the flavonoid glucuronides, while the aqueous fraction contained polar materials believed to be unidentified salts and amino acids/proteins.  Samples of relatively pure flavonoids could be obtained by further processing the flavonoid containing extracts on a reverse phase column using gradient water-methanol elution.
 
Flavonoids in Milk Thistle

In general, flavonoids are widespread in the plant kingdom but glycuronide derivatives are not the most common representatives – glycosides and aglycones are much more common.  Flavonoids are mostly anti-oxidants, considered to possess nutraceutical qualities, and by and large, generally recognized as safe  (GRAS status).   Apigenin glucuronides have previously been isolated from alfalfa leaves 2 and galacturonides identified in milk thistle flowers1.

Searching the patent literature afforded two3,4  patents which describe utilization of flavonoid glycuronides.  In one they were observed to promote improved solubility and uptake of various drugs; in the other they were used to prolong the life of natural anthocyanin pigments used as coloring agents. 

Fig. 2.2.1.        Putative structures of major (1) and minor (2) flavonoids observed in air-dried mature Silybum marianum leaves obtained from field plants, University of Saskachewan 2006/07.

2.2.2    Non-Flavonoid Components

Examination of the hplc-ms data (see Appendix 2.1) led to the tentative identification of triterpenes having a molecular weight of 456, possibly ursolic and oleanolic acids.  Previously reported5 oxygenated triterpenes were not observed.  The leaves are also considered a rich source of amino acids (both free and in proteins, (14%) as well as magnesium and potassium (source: http://www.ars-grin.gov/cgi-bin/duke/farmacy2.pl) and are considered edible on removal of the spiny thorns.  The bulk of the extract was the water soluble material which, based on the above report, was likely a mixture of salts, amino acids, proteins, and possibly polysaccharides.

2.2.3    Bioassays/Reported Activities

The major flavonoid (MW 446) was evaluated in hemolysis and apoptosis assays.  It was found not to be hemolytic in sheep red blood cells (HD50 >100 μM) and did not cause any increase in caspase 3/7 activity or mitochondrial pertabation in PC-3 human prostate cancer cells at 50 μM (Dual Sensor MitoCasp assay using flow cytometry).  Both of these assays suggest that the flavonoids have low cytotoxicity.  This is further corroborated by historical usage of the leaves as a tea:
 “The flowers and leaves of the milk thistle plant can also be used in an infusion. A milk thistle hot tea is used to stimulate milk production in nursing mothers, as well as to treat digestive problems.” (http://www.disability-resource.com/medical-health/alternative-medicine/herbal/antioxidant-rich-milk-thistle-herbal-remedy.php).  The above results in combination with reports of historical usage suggest low toxicity of the contained flavonoids.
Leaf extracts have been shown to extert anti-inflammatory properties in rats.7

2.2.4.   Conclusion: Potential Utility of Aerial Parts of Plant

Since the aerial part of the plant represents quite a large amount of biomass, suitable processing could lead to quantities of triterpenes (ursolic and oleanolic acids), potentially useful in cosmetic formulations (based on published anti-inflammatory properties), apigenin/luteolin glucuronides potentially useful in nutra- and /or pharmaceutical applications as outlined in the cited patents, and protein/amino acids potentially useful for food/feed applications.
 
For commercial development, nutraceutical claims for the flavonoids would have to be established and identification of salts and amino acids present in the aqueous fraction would be required.

Finally, development of milk thistle without thorns would be interesting – perhaps the plant could then be developed as a nutritious salad crop for human use or as a forage crop.  Allergy considerations may temper potential food uses.

Appendix:  2.1            HPLC-MS Data for Milk Thistle Leaf Extracts

Fig. 2.2.1.  HPLC profiles of extract obtained from air-dried mature leaves of milk thistle.  Top: uv detection @ 280-340 nm; Middle: uv detection @ 200-400nm; Bottom: mass detection (m/e 100 – 1900).

Fig 2.2.2.  UV spectrum of compound 1 and 2.

Fig. 2.2.3.  Mass spectrum (electrospray, positive ion) of compound 1, showing loss of galacturonate (or glucuronate).

Fig. 2.2.4.  Extracted ion  (m/z 456) mass chromatogram of MT leaf extract.  Peaks tentatively assigned as oleanolic and ursolic acids.

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Grading to Improve Milk Thistle Quality

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It has been established that the quality of milk thistle increases as the seed matures. Therefore the quality of a given seed lot can be improved if the seed can be sorted for maturity. Immature milk thistle seeds are small, thin and quite light in color by comparison with mature seed. Milk thistle seed cab be easily sorted with readily available equipment based on size and density. This does an acceptable job of sorting out the very immature seeds ... but cannot achieve any further grade improvement. It is possible however, to further sort based on seed color.

In 2003, we used a color sorter to grade a seed lot (Richter’s) and then examined the silymarin content of the resulting grade categories. There was some tendency for the silymarin content to increase with increasingly dark seed color. In 2004, we ranked the various cultivars tested in the germplasm evaluation trial for seed coat color (darkest = rank of 1) and silymarin content (highest = rank of 1)(Figure 1.7). We found that there was little direct association between seed coat color and silymarin content. We also ranked various lots of the cv. Richter’s grown in differing trials (* in Fig. 1.7). Again, there is little in the way of a relationship between coat color and silymarin content. This suggests limited potential to use color sorting technology to further improve sorting for quality in milk thistle.

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