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.

The Real Gumbo Recipe

I wanted to follow up requests in response to my post on Spices, and share the gumbo recipe I mentioned. 

It’s my mom’s from her mom, Nannie (Louise P. Curtis)

I’ve used Nannie’s original language

Start with 1 large TBS bacon, ham or sausage drippings in heavy saucepan

Chop 1 pound of okra, 1 large onion, 1 large bell pepper and 5 cloves of garlic

Fry in oil until wilted. Okra will lose stringiness.

Add 1-2 TBS of flour. Cook slowly about 30-45 minutes until okra gets drier.

Add 1 can tomato paste

Add hot seafood water, about 4 cups

Add seafood 1  lb crabmeat and 1 lb peeled shrimp

Add seasonings:

  • 2 drops Tabasco or cayenne pepper
  • salt and pepper to taste
  • a couple of bay leaves
  • chopped celery (1/4 cup)
  • green onions and parsley (optional)

Simmer for about 1 hour over very low fire

Serve over rice

 

Note: I always added olive oil and Louisiana Crystal Hot Sauce and opened a bottle of Jax or two.

 

Spices

I was lucky enough to grow up all over the world – Thailand, Hong Kong, Germany, Romania and Greece. Some of my fondest memories are around food. My mom was a fabulous cook, which was a vital part of my father’s work as a cultural affairs officer in the US Foreign Service.

Vanilla bean plant

One of my earliest memories was of making a landing in Anchorage Alaska on a return trip from Bangkok to my parents’ home town of Biloxi, Mississippi. I remember the large stuffed polar bear in the lobby, and more importantly, my first taste of vanilla ice cream. Having come from the heat of Bangkok, with my daily food rich in hot peppers and ginger, this cooling fragrant treat was nectar from the gods.

When I started to learn to cook standing over my mom’s pot of gumbo she stressed the importance of simmer with the last ingredients – two bay leaves. They looked so simple – small dried plant material –  but an hour later, the aroma of my Nannie’s gumbo was bigger than just the file and seafood, with the bay leaves providing a vibrant top note.  Years later, deep into a career researching active molecules in medicinal plants, I realized that the food culture I grew up in was full of medicinal plants in the spices added to our evening meals.

Bay Leaf

Spices both sense of taste and smell. Our sense of smell, in particular, is influenced by the characteristics of the molecules responsible for odor. How quickly do they volatilize (how easily molecules vaporize)? How soluble are they in water or oil?  How acidic is the food (a tomato based food or something with lemon)? Even our genes can influence the sense of smell.

A molecule that we can sense through smell is called a fragrance or odorant. Those molecules need to reach the olfactory system in the upper part of our nose. To do so, generally the molecular weight of these molecules needs to be equal to or less than 300 g/mol (a scientific designation for the mass of a substance divided by the amount of the substance). Ultimately, our sense of smell comes down to a pattern of activity of neurons in the brain responding to the stimulus of odor molecules binding to receptors (Malnic et al., 1999).

We have receptors (a protein in a cell membrane that responds to molecule attaching to it) in our nasal passages that are triggered by these molecules. Each olfactory receptor recognizes more than one odorant, and each odorant can be detected by several different olfactory receptors. This reflects a combinatorial process common to biological systems. Several factors influence the resulting signal sent from the receptor site.

We know that the shape of a molecule is important (Saberi and Seyed-Allaei, 2016), and that as the molecule binds (or sticks to) a receptor, the receptor changes shape, which leads to neural signals reaching the brain.  Several theories exist to help us understand how to map the process of detecting odors, based largely on chemical qualities of the odorants.

Mori et al. (1994) suggested that the odor signal are more complex than a single receptor binding event, rather the signal is a product of a series of receptor excitations from numerous receptor sub sites (odotypes). This became known as the Odotope Theory. This theory also explains odor-less molecules as the presence sub sites more numerous than the limit of potential binding sites.

A second important theory, Dyson (1938) originated the Vibrational Theory that Wright (1982) later refined. This theory posits that olfactory receptors might really sense vibrational energy on a quantum level rather than structural shapes of the molecule when detecting odors. Quantum mechanics describes nature at the smallest scales of energy levels of atoms or substances.

Combining the two theories, Turin and Yoshi suggest that the tightness of receptor binding, based on both the physical and charge shape (polarity) of an odorant molecule may influence the intensity of the odor, while the character of the odor is effected by vibrational characteristics of the molecule.

To help visualize the theories, we can take a series of molecules found in medicinal plants that have a common base structure, and explore how their odor characteristics may be influenced by chemical features such as solubility, volatility, molecular shape and size (see Table 1).

Starting with the basic structure, vanillin give vanilla its signature sweet, perfumed, woody aroma. The molecular weight is relatively low, and it volatilizes easily, filling a room with the odor when cooked. The oxygen R-groups (groups that “hang” from the ring) on the benzene ring of vanillin make it highly solubility in water.

Built from the same basic structure, eugenol has a short hydro-carbon tail that gives it a stronger odor than vanillin, the familiar aroma found in bay leaf or allspice. This hydro-carbon tail also makes it more fat soluble and may influence the intensity of receptor binding. The odor threshold of eugenol is also lower than that of vanillin. The less polar binding site on the molecule may influence the strength of binding in this example, and explain why the vanillin odor is not as persistent as that of bay leaf or allspice.

Table 1: Vanillin Based Molecular Structures with Odor Thresholds

Vanillin

(Odor Threshold: 20-200ppb)*

Eugenol

(Odor Threshold: 6-30ppb)

Zingerone

(Odor Threshold: na)

Capsaicin

(Odor Threshold: na)

*http://www.leffingwell.com/odorthre.htm

Ginger plant

Zingerone has an even longer hydro-carbon tail connected to the basic vanillin shape, making it insoluble in water. Found in ginger and mustard oil, it gives a rich, sweet, warm and woody fragrance. However, the presence of the carbonyl group (C=O) in the tail means that the zingerone molecules tend to attract each other, limiting how easily it volatilizes. Thus, ginger is less likely to fill a room as quickly with its aroma than vanilla.

Even though Capsaicin also has a long, hyrdo-carbon rich tail, the polar amide group (-NHCO-) makes it slightly more soluble in water than zingerone. The size of the tail also limits the molecule’s volatility. Capsaicin has no odor, and given the numerous sites along that long tail with the potential of binding to a receptor sub sites, this may be an example of a molecule with too many binding sites, as the Odotope theory suggests.

Chili peppers

So the next time in you are in the kitchen and the aromas are filling your senses, see if you can think about the shapes and characteristics of the molecules influencing that wonderful moment.

 

References:

  1. Malnic B, Hirono J, Sato T, Buck LB. (1999) Combinatorial receptor codes for odors. 96(5), 713.
  2. Saberi M, Seyed-Allaei H (April 2016). “Odorant receptors of Drosophila are sensitive to the molecular volume of odorants”. Scientific Reports. 6: 25103. doi:1038/srep25103.
  3. Mori, K. and Shepherd, GM. (1994). Emerging principles of molecular signal processing by mitral/tufted cells in the olfactory bulb. Semin Cell Biol 5-1:65-74.
  4. Dyson, G.M. The scientific basis of odour . Chem Ind 57: 647-651.
  5. Wright, R.H. (1982) The sense of smell CRCpress, Boca Raton, Florida, USA.
  6. Turin, L, and Yoshi, F. (2003) Structure-Odor Relations: a modern perspective in Handbook of Olfaction and Gustation Second Edition. Doty RL, Ed. New York Marcel Dekker.