Fermentation as an Extraction Method

Although most cultures have fermented food as a staple in their dietary patterns, little has been reported on the use of fermentation as an herbal extraction method. The process may contribute more than just modifying solvent pH. Rizzello et al. (2013) reported using a lactic acid fermentation with specific yeast strains that improved the antioxidant activity of Echinacea purpurea. They compared fermentation extraction to either a methanol or water extract without fermentation. The greatest antimicrobial activity was associated with low molecular mass compounds negated in the presence of digestive enzymes, suggesting small peptides as the active agent.  The authors cited other experiments with grapes, soy and cereal grains where the fermentation process increased bio-availability of  certain compounds and produced novel chemical species.

This process is worth exploring by both herbal supplement companies and herbalist as a new medicine making method. Traditional texts provide some guidance.  Enzymatic processes can optimize extraction of plant cellular content at lower temperatures. And research (Mishra et al., 2010; Mulay and Khale, 2011) applying traditional Ayurvedic methods of  fermented extraction found reduced toxicity in the final product. This opens up a little explored market around functional foods as well.

Reference:

  1. Mishra AKGupta AGupta VSand RBansal P2010Asava and arishta: an Ayrvedic medicine – an overview. Int J Pharm Biol Arch. 1(1):2430.
  2. Mulay SKhale A2011Asavarishtas through improved fermentation technology. Int J Pharma Sci Res. 2(6):14211425.
  3. Rizzello, CG et al. (2013) Lactic acid fermentation as a tool to enhance the functional features of Echinacea spp. Microbial Cell Factories. 12:44-59.

 

Not Blueberry Pie, but Close

Vaccinium myrtillus
Image via Wikipedia

Bilberry (Vaccinium myrtillus) contains varying levels of phenolic compounds – anthocyanins, chlorogenic acid derivatives, hydroxycinnamic acids, flavonol glycosides, catechins, and proanthocyanidins. Research by Martz et al (2010) elucidated how levels of bilberry leaf phenolics differed along an ecological gradient in boreal forests running north to south in Finland. These regions differ in latitude, altitude, over story cover, levels of continuous light, temperature and associated frost spells.

An analysis of bilberry leaves showed that major phenolic changes in bilberry leaves appeared in the first stages of leaf development. As important, synthesis and accumulation of flavonoids was delayed in the forest compared to the high light sites. Two-fold higher flavonoid levels appeared in leaf tissue growing in high-light intensity sites, higher latitudes, and/or higher altitudes compared to in lower altitudes and low-light intensity sites.

Close and Mcarther (2002) previously theorized that the presence of greater phenolic levels in leaf tissue found in northern regions was a response to colder temperatures, which would limit essential enzyme function, during periods of maximal photo-oxidative stress (Close and Mcarther, 2002).  However, Martz et al (2010) also showed that leaf flavanoid genes were highly expressed in shade, but that the timing of expression appeared to alter the relative metabolite levels in shade compared to sun exposed bilberry leaf.

Mudge et al., (2016), researched phenolic profiles of wild elderberry fruits (Sambucus nigra subsp. canadensis) over two years in eastern US, noting that flavanols (quercitin, isoquercitin, rutin) and chlorogenic acid metabolite concentrations were higher in the southeast, particularly interior. They suggested the variation of phytochemical profiles of the berries were impacted by genetic or environmental factors without understanding on which was more important.

What’s missing from the data picture includes a more complex measurement of ecological influences, such as response to herbivory and rhizosphere fungal associations? This type of whole community data would help to build a more complete picture of  plant response.

This requires sampling, sampling, sampling.

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  1. Martz, F., Jaakola, L., Julkunen-Tiitto, R. and Stark, S. (2010). Phenolic Composition and Antioxidant Capacity of Bilberry (Vaccinium myrtillus) Leaves in Northern Europe Following Foliar Development and Along Environmental Gradients. J Chem Ecol, published online, 19 August 2010
  2. Close, D.C., and Mcarther, C. (2002). Rethinking the role of many plant phenolics—protection from photodamage not herbivores? Oikos. 99:166–172
  3. Mudge, E., Applequist, W. L., Finley, J., Lister, P., Townesmith, A. K., Walker, K. M., & Brown, P. N. (2016). Variation of Select Flavonols and Chlorogenic Acid Content of Elderberry Collected Throughout the Eastern United States. Journal of food composition and analysis : an official publication of the United Nations University, International Network of Food Data Systems, 47, 52–59.

Ginseng Allelopathy in the Rhizosphere

The active, therapeutic phytochemicals in the Panax spp. appear to be ginsenosides that consist of an aglycone base structure and glycosides (sugar molecules). These compounds can be referred to as triterpene glycosides, triterpene saponins and steroid saponins. The varied nomenclature comes from the multiple ways of defining the molecule. Saponins froth when shaken. And from the image below, ginsenoside aglycones contain a steroid backbone. Based on the numbers of carbons they can also be classified as a triterpene. 

Ginsensodie Steroid

So what role do these phytochemicals they play on the plants behalf? Saponins as class have anti-fungal properties and may act allelopathically (Carter et al., 1999). The sterol portion of the molecule appears to be inserted into and disrupt fungal membrane integrity by interacting with fungal sterols present.

Nicole et al. (2002) noted that American ginseng saponins inhibit in vitro growth of the fungus Trichoderma spp., but stimulate growth of Cylindrocarpon destructans.

In a followup study to  investigate the concentration of ginsensosides in rhizosphere, Nicol et al. (2003) collected ginsenosides from root associated soil several times between 1999-2002. They found that the concentration in the soil ranged 0.02 – 0.098%. They also collected root exudate from pot-grown ginseng over 22 days, using an exudate trapping system,  which yielded a concentration of 0.6% ginsenosides. We need to test whether this soil concentration level can be considered an active allelopathic level.

Additional evidence supporting the sterol disruption hypothesis can be found in the Pythiaceous fungi (especially Pythium spp. and Phytophthora spp.) that lack sterols in their  cell walls.  Growth of these fungi appear to be stimulated by both the presence of fungal sterols (ergosterol) and ginsenosides in the medium of in vitro studies. The authors suggested potential mechanisms include: 

The ginsenosides…

  • provide a carbon sink for the fungus.
  • alter fungal membranes in a positive manner.
  • act as a fungal growth hormone.

Future studies should look at the ratio of ginsensosides and their respective influence on fungi.

References:

  1. Carter, JP, Spink, J, Cannon, PF, Daniels, and Osbourn, AE. (1999) Isolation, Characterization, and Avenacin Sensitivity of a Diverse Collection of Cereal-Root- Colonizing Fungi.AppliedandEnvironmentalMicrobiology. 65(8): 3364–3372.
  2. Nicol, RW, Traquair, JA and , Bernards, MA. (2002) Ginsenosides as host resistance factors in American ginseng (Panaxquinquefolius).CanadianJournalofBotany. 80(5): 557-562.
  3. Nicol RW, Yousef L, Traquair JA, and Bernards MA. (2003) Ginsenosides stimulate the growth of soilborne pathogens of American ginseng.Phytochemistry. 64(1):257-64.

Rhizosphere Influence on Plant Medicine

Einjähriger Beifuß (Artemisia annua)
Artemisia annua
Wikipedia

Mycorrhization leads to nutrient and information flow, often in both directions. The plant root supplies sugars to the fungus, while the fungus induces Jasmonic Acid biosynthetic enzymes in the plant, leading to an increase in jasmonate ­ levels that enhance the accumulation of soluble sugars in plant root and the production  of plant root defense compounds.

From a research article,  the presence of mycorrhizal fungus, Glomus mosseae and nitrogen fixing Bacillus subtilis on the roots influenced the levels of plant biomass growth, and the yield of an important medicinally active phytochemical, artemisinin, from Artemisia annua L and used as an anti-malarial treatment.

Gabriele et al. (2016) investigated the effect of mycorrhizal soil inoculation of various Sangiovese wine grapes and found the presence of the fungus increased levels of 14 polyphenols compared to un-inoculated plants. Here the presence of symbiotic relations in the soil altered the phytochemical makeup of fruit.

So how are the plant roots attracting mycorrhizal symbionts? Plant produced flavanoid compounds accumulate at root tips/cap and make up a large portion of root exudate (the portion of the root sap excreted to the external environment). These phytochemicals are easily modified and their biosynthesis is triggered  by transcription factors, which suggests a role as elicited signal compounds – compounds that are made specifically in response to conversation from rhizosphere fungi and bacteria. Interestingly, their presence in the rhizosphere soil triggers mycorrhizal fungi to explore their surroundings (Hassan and Mathesius, 2012), perhaps increasing the likely hood of contact with plant roots.

Given the high price of American wild grown ginseng, the ecological influence on ginsenoside formation, and ultimately, the therapeutic value, points to optimizing the rhizosphere cross talk by way of forest farming.

The highest ginsenoside content occurs (from highest to lowest) in the root hairs > lateral roots > cortex > interior taproot (Li and Wardle, 2002), exactly where we should expect a chemical conversation to occur.

Within this class of compounds we designate as ginsenosides, two molecular forms are dominant, protopanaxadiols and protopanaxatriols. Data from two different papers (Zhu et al., 2004: Wang et al., 2010) compared levels of diols and triols in different species and sources of ginseng. American ginseng (Panax quinquefolia) had higher levels of the triols (especially Rg1) compare to Chinese ginseng (P. ginseng), which had higher levels of diols (especially Rb1  Rd).

Structures-of-ginsenosides-from-Panax-ginseng-Glc-glucose-Rha-rhamnose-Araf
Li, H, Lee, JH, and Ha, JM. (2008) Effective Purification of Ginsenosides from Cultured Wild Ginseng Roots, Red Ginseng, and White Ginseng with Macroporous Resins. Journal of Microbiology and Biotechnology. 18(11):1789-91. DOI: 10.4014/jmb.0800.192

Comparing wild grown versus cultivated plants within each species, a similar pattern emerged, with wild plants showing a higher concentration of triols (especially Rg1  Re), while cultivated plants had higher concentration of diols (especially RbRb2).

James, et al. (2013) investigated levels of diols and triols in wild sourced P. quinquefolia leaf and root  in a North Carolina collection, finding that there was no relationship between age and ginsenoside content. However total ginsensosides were higher in the leaf, as was Rb2 and Rd (diols), In the root tissue, Rb1(diol) and Rg1 (triol) was found to be higher.

This has implications for how we “farm” medicine and speaks to a long held tenet; complex interactions in native ecologies, including the soil,  produce medicinal plant crops that are more biologically active. Farm versus wild grown ginseng is only one example. What’s been your experience as a imbiber, herbalist, researcher, plant grower or manufacturer?

 

Lousewort

Watercolor painting of Pedicularis_bracteosa-2.
(Wikipedia)

Pedicularis bracteosa
P. canadensis
P. greenlandica
P. attollens

Ecology – Hemi-parasite

Molecular phylogeny more recently placed the genus Pedicularis in the Orobanchaceae, when formally it had been  in part of Scrophulariaceae.

Herbalist Michael Moore has written on the therapeutic uses Pedicularis spp., as has David Winston and 7Song.

The plant is an excellent skeletal muscle relaxant, with some of its specific indications as follows:

  • Adrenaline-stressed or nerve impinged muscles
  • Hypertonicity and muscular rigidity
  • Children with highly excited flight or fight response

I’ve created formula with Pedicularis for massage therapist and chiropractors to  increase “hold” of treatment. In particular, it combines well with other skeletal muscle relaxants include Black cohosh (Actaea racemosa), Kava kava (Piper methysticum) and Skullcap (Scutellaria).

Since it is a root parasite the plant can take up compounds from it’s host plant. Schnieder and Stermitz (1990) noted that several Pediculars.spp. uptake alkaloids from a variety of hosts: pyrrolizidine alkaloid senecionine from Senecio triangularis, anagyrine from Thermopsis montanaN-methylcytisine from  Thermopsis divaricarpa and quinolizidines from Lupinus argenteus.

For this reason it’s unclear which therapeutic compounds are made by the plant and which come from host, which can make the safety profile a little trickier to predict. The host compounds can even alter the pigment of Pedicularis flowers. Best to find it growing alone in its own stand, or rely on a highly skilled wildcrafter to help identify a good stand.

Experimentation and observational studies have shown that two hosts can be parasitized simultaneously. Such threesomes seem to improve the overall growth performance and survivability of the parasite.

This is a fascinating plant that requires the deft touch of an herbalist, with science providing interesting data on how plant parasites interact with their ecosystem.

 

 

 

 

Plant Chemical Out Posts

Flower of Garden Strawberry (Fragaria ×ananassa)
Image via Wikipedia

The search for chemical mediators in plant root rhizosphere interactions with symbiotic and pathogenic organisms found in the soil continues to generate interesting research. Martha Hawes group at the University of Arizona reported on the role of sugars, proteins and small molecules found in root cap secretions – a mucilaginous mixture that covers the growing root tip and “converses” with the surrounding matrix of living organisms. The cap is rich in root border cells, which detach from the growing root tip. Curlango-Rivera et al (2010) provides us a bit more detail about which metabolites are biologically active. Neither sugars nor amino acids triggered root growth or border cell production. Transient exposure to biologically active concentration levels of the isoflavonoid pisatin, a phytoalexin, stimulated root border cell production but not root tip growth. I wonder if inhibition of root elongation may “reset” plant growth patterns as root border cells, acting as chemical sense organs, define the nature of the environment?

A second paper used histochemcial methods to profile root metabolites in plants from the Rose family (Hoffman et al., 2010). They found flavan-3-ol molecules in the root tip and border cells. Their findings suggest that the distribution of flavan-3-ols in Fragaria and Malus is under tight developmental control. These molecules are found in plants as catechin and epicatechin derivatives and in long chain (polymeric) form. They influence the taste and medicinal potential of green tea and wine, to name a few well-known plants. Previous researchers summarized their role in chelating toxic cations (metals) in the soil, establishing mycorrhizal interactions and priming plant root defense. This paper suggests a role in the transport of the long distance plant hormone auxin, which would link the chemical cross talk at root border cells with responses that occur in tissue distal to root tips. Hoffman’s research lacked a clear distinction of whether the monomeric or polymeric flavan-3-ol forms where the active species. This has plagued plant research for some time, since the analytical methods for detecting the polymeric forms have been crude and ineffective. All of their samples were from a botanical garden. I wonder if the flavan-3-ol profile would differ compared to native wild grown species?

References:

  1. Curlango-Rivera, G. et al. (2010) Plant Soil 332:267-275
  2. Hoffmann, T. et al. (2011) Plant Biology, 13: no. doi: 10.1111/j.1438-8677.2011.00462.x

Good Wine, Good Fungi

A study of organic soils found that the those associated with organic gardening compared to conventional methods or native grasslands, was very similar in types and diversity of mycorrhizal fungal taxa to that of the native soils. Increasingly, viticulturalists have been promoting the sustainability of using organic techniques over the fungicide heavy approaches of conventional wine management practices, and that this fundamental investment in “terroir” makes better wine. One method is to restore the density and diversity of beneficial, symbiotic fungi in the vineyard soil. These fungi are seriously depleted in soils that have had extensive chemical fertilizers, fungicides or pesticides applied.

Mycorrhizal inoculum applied to new vines plantings and as a dressing to cover crop used to improve nitrogen availability in vineyard soils, associates with the vine roots and  increases both the available levels of organic carbon and the water holding capacity of the surrounding soils. And with healthy vines, and a biological approach to vineyard management in place, the rhizosphere community rich in mycorrhizal fungi can influence the quality of wine produced. 

Gabriele et al. (2016) investigated the effect of mycorrhizal inoculation of various Sangiovese wine grapes. The symbiotic relationships improved the oxidative stability, thus the potential ability of the wine to age, and increased 14 polyphenols compared to un-inoculated plants. The later effect may improve the structure and the flavor profile of the wine.

I’ve asked to join the downstream portion of the research team to investigate the impact of these changes on the consumers experience.