Anna-Marija Helt, PH.D.

Osadha Natural Health

Herb Notes

Old fashioned ways to identify poisonous mushrooms (if you want a trip to the ER or the morgue, that is)

Deadly Amanita mushrooms will test as “safe” for most of these. In other words, DO NOT use these to suss out which mushrooms are poisonous….

From the upcoming “Seriously Poisonous Mushrooms of North America” Zoom class

I wouldn’t use these methods if I were you.

A mushroom is toxic if:

…a silver coin blackens if placed in water with the mushroom    

…the mushroom curdles milk

…it’s cap skin is difficult to remove

…it turns a clove of garlic blue or black when cooked together

…if it grows on dung (the ubiquitous grocery store Button Mushroom would be flagged 

    as toxic)

…if its flesh turns a different color upon slicing

…if its odor is unpleasant

Cannabis Endophytes: An answer to a question

Originally published in Plant Healer Quarterly

When teaching about endophytes at the Good Medicine Confluence this past May, someone wanted to know about Cannabis endophytes. I didn’t know anything about that topic other than that all plants tested thus far have endophytes.  I decided to look into it some more, and here we go with an up-to-date view….. 

Endophytes 101

In case you missed the class and accompanying essays…a quick review. 

Endophytes are fungi and bacteria that live inside of plants (hence, endo “inside” and phyte “plant”).  They either live packed in between or within the plant’s cells. This distinguishes them from epiphytes, which live on the surface of the plant. 

Endophytes have been hitching a ride in plants for a very long time. They may have jumped on board shortly after plants appeared on the planet, based on evolutionary data and the fossil record.  This long association implies that plants and endophytes have, possibly to a large extent, directed each others’ evolution.  Indeed, the genes of a plant’s resident endophytes provide the plant with a larger toolbox for dealing with changing conditions.  

As an example…   A Pine tree and its descendants may take a long time to accrue beneficial mutations in their genes…not quickly enough to survive, say, an infestation by a parasitic beetle.  But, the more rapid response of the Pine’s individual endophytes as well as the endophyte population structure as a whole may provide the means for an individual tree to adapt better to changing conditions. This means that the tree will be around to pass its own genes on, but also to pass on at least some of its endophytes. (Endophytes are found in seeds as well as in other plant parts, and in the surrounding soil, and may even spread via spores).  Same idea with us and our microbiome:  We essentially get some of the benefits of “our” microbial genes.

Along these lines, endophytes may benefit the host plant in several ways. For instance, endophytes may produce metabolites that deter grazing by insects or animals. Or, may protect the plant against infection by pathogenic microbes.  Some endophytes enhance plant development and/or growth, or may inhibit the growth of the plant’s competitors. 

However, endophytes don’t simply show up at the plant’s door and say “Hey, how ‘bout we come on board and help you out?”.  In fact, some endophytes may be parasitic, taking from the plant but not giving much back in return.  In any event, the endophyte gets a home along with nutrients. This arrangement is similar to ours with our resident flora…not all of the bugs in our microbiome are necessarily to our benefit, though many are.

Cohabitation of endophytes and plants is relevant to us as herbalists.  More specifically, some of what we call “plant medicine” is actually plant and endophyte medicine, or even largely endophyte medicine.  Why is this?  Because endophytes influence the secondary metabolites (the “medicine”) present in a plant.  Some endophytes do this by stimulating the plant to increase synthesis of a particular plant metabolite. This is seen with the synthesis of Echinacea’s anti-inflammatory alkylamides (1, 2). Others trigger the plant to produce a chemical that wouldn’t be there in the absence of the endophyte (eg. resveratrol in Douglass Firs) (3).

Weirdly enough, sometimes both the endophyte and plant make the same chemical (for example, paclitaxol in the case of the Yew tree and its endophytes (3). Finally, some endophytes generate compounds that the plant itself doesn’t make (so cool!). For instance, endophytes in Guduchi make inhibitors of xanathine oxidase, which is involved in uric acid production (Guduchi is used for gout…)(4).  

On to Cannabis

Does it really need an introduction?  

Cannabis is getting a lot of attention for its medicinal properties beyond its psychoactive effects.The medicinal chemistry of Cannabis is super interesting.  The secondary metabolites number over 400, including cannabinoids, terpenoids, flavonoids and lignans that have a wide range of activities. Many Cannabis constituents — terpenes such as ?-myrcene and limonene, for example — are also found in a slew of other medicinal plants. 

Cannabinoids as a chemical class were originally identified in Cannabis (no duh). Technically speaking, cannabinoids are “terpenophenolic” compounds, with terpenes consisting of isoprenyl units.  Chemicals in the pytocannabinoid family are relatively limited in nature, but are found in a number of plants beyond Cannabis. Helichrysum umbraculiserum, from Africa, is reported to contain cannabigerol (CBG) and its precursor acid form CBGa, along with  other chemically-related compounds (5). CBG is the precursor to other cannabinoids such as cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN) and the rest of the 3-lettered collection.  That said, the other cannabinoids aren’t found in Helichrysum, so it may lack the necessary enzymes for conversion.    Metabolites structurally related (but not identical) to CBG, CBD, THC, and cannabichromene (CBC) are synthesized variously by Rodadendron, Liverwort, American Licorice and other plants, and even in a fungus, Cylindrocarpon olidum (5). 

Cannabis and endophytes

Now, we’re finally getting to the point of the article!  Cannabis has both fungal and bacterial endophytes, with microbial species and population structure varying based on cultivar (and also both  “hemp” and “marijuana”), growth region, growth stage and plant part (6, 7). 

Multiple fungal species from Aspergillus, Penicillium, Chaetomium, Alternaria and others have been isolated from Cannabis, mainly from cultivated plants though not entirely (8-11), and the bacteria genera isolated include Bacillus, Staphylococcus, Pseudomonas, Acinetobacter, Enterobacter, Pantonea and others.  (7, 11,12).  Endophytes live in the leaves, petioles, twigs, buds and roots (6, 10, 12). 

Endophytes and cannabinoid production

Aside from someone asking me about Cannabis endophytes at the Good Medicine Confluence, the other reason I embarked on this article was to nerd out on how endophytes influence cannabinoid production. 

And……..not.   Not that endophytes don’t influence the presence of cannabinoids in Cannabis. There simply are no published reports documenting this that I’ve found via either Medline, Ovid or Google Scholar databases.  Plenty of research groups have speculated that endophytes do, indeed, influence cannabinoid production.  But, again, no actual reports, as yet, that I know of.  Disappointing!  But  I’m going to out on a limb and guess that more than one research group is working on this and that reports will likely come out over the next year or so, so stay tuned.  

I’m not sure if the issue is in technical challenges?  Cannabis endophytes have been identified starting not quite a decade ago….     To truly nail how the endophytes associated with a particular plant species, the plant actually has to be grown under sterile conditions and then inoculated with the endophytes. (This has been done, for instance, with Echinacea where plants were grown in lab conditions from seeds sterilized of all microbes.)   I wonder if there is some difficulty in doing this with Cannabis for some reason not immediately obvious to me, having no experience in growing it…. 

Endophytes and plant growth

OK, so I struck out on the endophyte-cannabinoid thing.  Next was looking into how endophytes may influence Cannabis growth, given that endophytes are known to improve the growth, and hence, yield, of other medicinal plants.  And, again, not a hell of a lot of work’s been published on this as yet.  What I could find:

There is a study from China reporting increased production of Hemp fiber in response to 6 Cannabis endophytes, including fungi in the Fusarium and Chaetomium genera, though I couldn’t access anything but the abstract and, thus couldn’t look at the methods or actual data (13).   

Endophytes isolated from Cannabis cultivars have been found to produce, when grown in culture, metabolites that when added to other plants increases growth. For example, endophytes from wild Cannabis increased the growth of canola (rapeseed) and increased the plants’ resistance against various types of stressors  enhance the growth of canola and also protect against various stress-inducing factors  (14).  Similarly, inoculation of rice plants with the Cannabis-derived endophytic fungus, Bipolaris sp. CSL-1 improved plant growth and chlorophyll content by producing 2 classes of plant hormone while also stimulating production of plant hormones by the plants (15). 

Flipping this approach around, microbe(s) not isolated from Cannabis increased were able to increase Cannabis plant height, stem basal area and bud size (16). Yield of product was increased by 16.5%  No idea what the bugs actually were, apparently it’s proprietary (“Mammoth P™”)(16).

All of this implies that the native endophytes in Cannabis may improve its growth, but, again, this hasn’t yet been demonstrated, to my knowledge. (At least I haven’t seen it in the research databases I’ve searched.)  Will put money on this data coming out soon as well.  

Endophyte-mediated protection from Cannabis pathogens

Finally, an area where there is a bit more information!  Cannabis endophytes may have a role in protecting the plant from pathogenic microbes.  The battle of the bugs, as it were. Which isn’t particularly surprising…this has been seen for other plants, and our microbiome does the same for us. 

Anyone who’s grown Cannabis knows it’s susceptible to infection by a wide variety of pathogenic microbes, including the fungi Botrytis cinerea (brown rot); Trichothecium roseum (a big problem in greenhouse-raised plants); Penicillium species (bud rot); Fusarium species (leaf and stem wilt) and even a protist, Phythium (root rot) (17, 18). 

Cannabis is also susceptible to mold and powdery mildew (17, 18). It’s been said by a Hemp expert named Dewey in the early 1900s that hemp has no enemies, but it turns out to be not so accurate…particularly in the green house. 

Studies done in culture have shown that Cannabis endophytes have activity against the 2 major Cannabis pathogens I just mentioned, Botrytis and Trichothecium (19) and against a passel of other plant pathogens (10, 20). One way Cannabis endophytes inhibit other microbes is by producing anti-microbial metabolites toxic or otherwise inhibitory to them.  

An even sneakier way Cannabis endophytes out compete pathogenic bugs is by rendering the pathogens harmless (21). This is accomplished by inactivating the pathogen’s virulence factors…the very things that make them a pathogen to the plant.  More specifically, some endophytic bacteria of Cannabis appear to disrupt quorum sensing by Cannabis pathogens (21), in a process known as “quorum quenching”.  Quorum sensing is a way that single celled organisms such as bacteria can behave somewhat like a multicellular organism by sending out chemical signals that regulate, as a group, activities such as replication, virulence, biofilm generation and antibiotic resistance.  This is good news for the plant. Keep in mind that it’s not that the resident endophytes are being nice to the plant, they’re simply protecting their home and meal ticket.

Is the presence of endophytes in Cannabis a risk factor for us?

While endophytes, by definition, don’t cause disease in their host plant, you may have noticed that some of the same genera that are Cannabis endophytes are also Cannabis pathogens.  Just to make things confusing, some Cannabis endophytes may be pathogenic to us.    

For example, representatives from the Aspergillus genus are endophytes in Cannabis (and other plants), and there are a number of cases where immunocompromised folks smoking Cannabis have developed dangerous lung infections with the fungus (22, 23). In some cases resulting in fatalities. There is also an immunocompetent person who developed aspergillosis after smoking Cannabis (23).   

Other fungal species, such as Fusarium, found in Cannabis can produce a variety of mycotoxins (22, 23).  Potentially toxic (to us) fungi native to Cannabis flowers include not just Apergillus, but also species in the Penicillium genus such as P. citrinium and P. paxilli  (23). Though this study didn’t distinguish endophytes from those fungi residing on the surface of the plant. 

To be clear, this issue isn’t limited to Cannabis…many food crops harbor endophytes that may not be great for us (24), so while the topic of plant endophytes and their benefits for our plant medicine is fascinating, it’s not entirely sunshine and roses for us.   

A handful of remaining questions on cannabis endophytes…

  1. Do Cannabis endophytes impact the synthesis of secondary metabolites such as cannabinoids, terpenoids, flavonoids and such in Cannabis?

Presumably the answer is “of course they do”.  Just waiting on the studies looking at this…. 

2.  Are there endophytes in Cannabis capable of synthesizing cannabinoids, and if so, are they able to do this in culture outside of the plant?  

While many of us herbalists prefer crude extracts, you know that some company somewhere is looking at this, either for pharmaceuticalization of individual cannabinoids and using them as source material for the generation of other cannabinoids.  

3. How much is the regional and strain variation in Cannabis chemistry impacted by their particular collection of endophytes?

It’s known that the endophyte populations of a particular plant species do vary based on region. For example, endophyte diversity is higher in equatorial regions compared to temperate regions.  This variation in endophyte species and population structure seen in different regions may be one of the reasons the medicine from a plant varies regionally. 

4.  Can Cannabis endophytes isolated from the wild and inoculated into cultivated strains to improve yield and cut down on resource utilization?

This is a question that almost certainly being looked at as well.  We know that in other medicinal plants, certain endophytes improve factors such as plant growth, resistance to pathogens and other stress and increased yield of medicinally-relevant metabolites.  Likely this will be the case for Cannabis as well. 

So, there it is. Cannabis endophytes in nutshell. To be continued as more research comes out… 

References

  1. Maggini, V, et al (2017) Plant-endophytes interaction influences the secondary metabolism in Echinacea purpurea (L.) Moench: an in vitro model. Scientific Rep. 7:16924. https://www.nature.com/articles/s41598-017-17110-w 
  2. Gualandi, RJ Jr. (2010) Fungal endophytes enhance growth and production of natural products in Echinacea purpurea (Moench.). ” Master’s Thesis, University of Tennessee. https://trace.tennessee.edu/utk_gradthes/713/
  3. Owen, NL & N Hundley (2004) Endophytes — The chemical synthesizers inside plants. Science Progress. 87(2):79-99. https://www.jstor.org/stable/43423175?seq=1#page_scan_tab_contents  
  4. Kapoor, N & S Saxena. (2018) Endophytic fungi of Tinospora cordifolia with anti-gout properties. 3 Biotech. 8(6):264.  https://www.researchgate.net/publication/325254828_Endophytic_fungi_of_Tinospora_cordifolia_with_anti-gout_properties
  5. Hanus, LO, et al (2016) Phytocannabinoids: A unified critical inventory. Nat Prod Rep. 33:1357-92.  https://pdfs.semanticscholar.org/fb32/29608775e567d718c6b80222b5ff23b90f90.pdf?_ga=2.189307310.423647943.1564852638-340685132.1558650599 
  6. Winston, ME, et al (2014) Understanding Cultivar-Specificity and Soil Determinants of the Cannabis Microbiome. PLoS ONE 9(6): e99641. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099641
  7. Backer, R, et al (2019) Closing the yield gap for Cannabis: A meta-analysis of factors determining Cannabis yield.  Front Plant Sci. 10:495.
  8. Zubek, S, et al (2012) Fungal root endophyte associations of medicinal plants. Nova Hedwigia. 94(4):525-40.  https://www.researchgate.net/publication/266577867_Fungal_root_endophyte_associations_of_medicinal_plants
  9. Kusari, P, et al (2013) Endophytic fungi harbored in Cannabis sativa L.: diversity and potential as biocontrol agents against host plant-specific phytopathogens. Fungal Diversity. 60:137-51. https://www.researchgate.net/publication/257800081_Endophytic_fungi_harbored_in_Cannabis_sativa_L_Diversity_and_potential_as_biocontrol_agents_against_host_plant-specific_phytopathogens
  10. Guatam, AK, et al (2013) Isolation of endophytic fungi from Cannabis sativa and study their antifungal potential. Arch Phytopathol Plant Protection. 46(6):627-35. https://www.tandfonline.com/doi/abs/10.1080/03235408.2012.749696
  11. Scott, M, et al (2018) Endophytes of industrial hemp (Cannabis sativa L.) cultivars: identification of culturable bacteria and fungi in leaves, petioles and seeds. Can J Microbiol. 64(10:664-80. https://www.ncbi.nlm.nih.gov/pubmed/29911410 
  12. Kusari, P, et al (2014) Quorum quenching is an antivirulence strategy employed by endophytic bacteria. Applied Microbiol Biotechnol. DOI 10.1007/s00253-014-5807-3. https://www.researchgate.net/publication/262537607_Quorum_quenching_is_an_antivirulence_strategy_employed_by_endophytic_bacteria 
  13. Jin, X, et al (2014) Effects of Endophytic Fungi Re-inoculation on Physiological and Agronomic Characters of Hemp (Cannabis sativa). Plant Diversity. 36(1):65-69. http://journal.kib.ac.cn/EN/abstract/abstract3287.shtml
  14. Azfal, I, et al (2015) Selective isolation and characterization of agriculturally beneficial endophytic bacteria from wild hemp using canola. Pak J Bot. 47(5):1999-2008. https://www.researchgate.net/publication/283086917_Selective_Isolation_and_Characterization_of_Agriculturally_Beneficial_Endophytic_Bacteria_from_Wild_Hemp_using_Canola
  15. Lubna, SA, et al (2019) Growth-promoting bioactivities of Bipolaris sp. CSL-1 isolated from Cannabis sativa suggest a distinctive role in modifying host plant phenotypic plasticity and functions. Acta Physiologiae Plant. 41:65
  16. Conant RT, et al(2017) Effects of a Microbial Biostimulant, Mammoth PTM, on Cannabis sativa Bud Yield. J Hortic 4: 191. https://link.springer.com/article/10.1007%2Fs11738-019-2852-7
  17. Punja, ZK (2018) Flower and foliage-infecting pathogens of marijuana (Cannabis sativa L.) plants. Can J Plant Path.  40(4). 
  18. Miller, M (2019) Pathogens Causing Wilting in Field-grown Cannabis Identified. Analytical Cannabis. https://www.analyticalcannabis.com/articles/pathogens-causing-wilting-in-field-grown-cannabis-identified-311392
  19. Kusari, P, et al (2017) Cannabis Endophytes and Their Application in Breeding and Physiological Fitness. Cannibis sativa L.- Botany and Biotechnology. https://www.researchgate.net/publication/318013755_Cannabis_Endophytes_and_Their_Application_in_Breeding_and_Physiological_Fitness
  20. Qadri, M,et al  (2013)  Identification and bioactive potential of endophytic fungi isolated from selected plants of the Western Himalayas. Springer Plus. 2:8.  https://springerplus.springeropen.com/articles/10.1186/2193-1801-2-8
  21. Kusari, P, et al (2014) Quorum quenching is an antivirulence strategy employed by endophytic bacteria. Appl Microbiol Biotechnol. DOI 10.1007/s00253-014-5807-3. https://www.researchgate.net/publication/262537607_Quorum_quenching_is_an_antivirulence_strategy_employed_by_endophytic_bacteria?enrichId=rgreq-328473c72c5df7173bcb39b8a31bdaaf-XXX&enrichSource=Y292ZXJQYWdlOzI2MjUzNzYwNztBUzoxODg2ODk1NjUxMDIwODBAMTQyMTk5ODUwNzAxMQ%3D%3D&el=1_x_2&_esc=publicationCoverPdf 
  22. McKernan, K, et al (2016) Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers. F1000 Research. 4:1422. http://f1000research.com/articles/4-1422/v2 
  23. Wielgusz, K & L Irzykowska (2017) Occurrence of pathogenic and endophytic fungi and their influence on quality of medicinal plants applied in management of neurological diseases and mental disorders. Herba Pol. 63(4):57-69. https://www.researchgate.net/publication/324914497_Occurrence_of_pathogenic_and_endophytic_fungi_and_their_influence_on_quality_of_medicinal_plants_applied_in_management_of_neurological_diseases_and_mental_disorders 
  24. Bouakline, A, et al (2016)  Fungal Contamination of Food in Hematology Units. J Clin Microbiol. doi: 10.12688/f1000research.7507.2 https://jcm.asm.org/content/38/11/4272

Endophytes – The Sequel. Your herbal medicines are more complicated than you may know!

Originally published in Plant Healer Quarterly

Endophytes are little critters that live either inside of or squished in between plant cells, and they’re a reminder that no organism, whether plant, person or platypus, is an individual. Rather, we’re communities of our own cells along with enormous numbers of resident microbes. We even have microbial DNA integrated into our chromosomes. And these bacterial, fungal and other critters themselves have their own collection of even smaller critters, namely viruses, within them.  Fractal nature…as above, so below. 

It’s clear that these little tag-alongs — endophytes, the gut microbiome, etc — have a big impact on health in us and in plants, and most likely in platypuses as well. (By the way, did you know that the correct plural form is actually platypodes? Who knew??).  “Endophyte” the word came from a 19th century mycologist, H. A. de Bary, to distinguish organisms living within a plant’s tissues from those that live on the plant surface  (“epiphytes”).  He was good with words, coming up with “symbiosis” as well (derived from Greek for “living together”). For more background on the what and potential whys of plant-endophyte cohabitation, visit the fall issue of Plant Healer Magazine. 

A bit of a side note, we know that many mushrooms are intimately linked with the roots of plants…these are “mycorrhizal” fungi and their relationship with plants is mutually beneficial.  But some mushrooms can live as endophytes, meaning completely inside of the plant.  For example, Turkey Tails inside of Ashwagandha and Waxy Caps inside of Plantain.  This was news to me!

So, endophytes and herbal medicine…  Endophytes have been found in every plant species tested so far, including medicinal plants. When you harvest some Plantain, for example, most of what you’ve got is obviously plant. But, endophytes are there in enormous numbers, even if they don’t weigh much. And they may be having a significant influence on what winds up in your tincture bottle.  How?  Endophytes can influence what secondary metabolites are present in the plant and at what levels.  These are the bits of the plant that constitute herbal medicine…flavonoids and other polyphenols, alkaloids, steroidal saponins, tannins, glycosides and such. 

Endophytes influence secondary metabolites in different ways.  Some endophytes produce metabolites that the plant itself doesn’t make. Some trigger the plant to produce metabolites that would otherwise not be present or would be there only there in low levels. Other endophytes synthesize metabolites that the plant also makes.  Endophytes, in some cases, can increase plant size and influence plant physiology in other ways. Selfishly for us herbalists, this can translate to more medicine from one plant!

Let’s look at the potential roles endophytes play in some of our best loved “herbal” medicines. 

Echinacea (Echinacea purpurea)

We all know that Echinacea polysaccharides stimulate the immune system, right?  Are you sure about that? 

Echinacea purpurea

It seems that a big part of Echinacea’s immune stimulation is directly attributable to endophytic (and surface) bacteria.  More specifically, bacterial lipopolysaccharide (LPS) and lipoproteins are the culprits.  These are actually not the secondary metabolites I’ve been blabbing about but are structural components of bacterial membranes.  Anyway, my first thought was “WTH????” after reading that bacterial stuff is largely responsible for Echinacea’s immune activation.  And, Echinacea is loaded with endophytes. One study alone isolated over 150 endophytic bacterial species (1), and this isn’t even considering how many are there but not able to be grown in culture or how many fungal species there are. 

How did folks decide that bacteria are important for Echinacea’s immune stimulation?  One way was to grow Echinacea plants from seeds that had been sterilized of any endophytes. Ethanolic extracts from the resultant endophyte-free Echinacea plants were not active in stimulating macrophage activity, while the control plants containing bacterial endophytes were active (2).  Macrophages are important cells in our innate immune response, and the ability to activate macrophages in culture is a common way to assess immune stimulation by plants, drugs, etc.  When I first read this, I figured it didn’t eliminate Echinacea polysaccharides as important contributors to the plant’s immune stimulating activity, since the extracts studied were made with ethanol and largely lacked plant polysaccharides. And, I stuck my tongue out and blew a raspberry in the general direction of the researchers (North Carolina). 

But, research with polysaccharides extracted from Echinacea also found that inactivating bacterial LPS and lipoproteins in the extracts destroyed most, though not all, of the immune stimulating activity (3).  This was true not just in the standard macrophage assay in a dish, but in vivo as well (3). Even in column purified polysaccharide preparations, bacterial lipoproteins are present (3), and it really doesn’t take much of either LPS or lipoprotein to trigger an immune response. Endophytes were sources of LPS and lipoproteins, though some may come from surface-contaminating bacteria as well (3). And these observations weren’t limited to Echinacea…similar results were seen with Black Walnut, American Ginseng, Alfalfa and others (3). Gah!!!

More along these lines…  When Echinacea preparations from different growers were assessed, they varied in levels of immune stimulation by about 100-fold, and this variation in activity was closely tied to the level of bacterial LPS and lipoproteins in the preparations rather than, for instance, the method of plant processing (4). And it’s not just any old bacterium that is responsible. Even though overall numbers of endophytic bacteria in Echinacea preparations correlated with how robust the immune stimulation was, particular bugs, such as Proteobacteria, were linked with the extent of immune stimulation (1, 2).

Alas, all is not lost for the polysaccharides. There is still residual immune stimulation when the LPS/lipoproteins are inactivated that could be due to the polysaccharides. Also, components of the immune system not evaluated in these various studies may be triggered by the polysaccharides.  In any event, the story is maybe a bit more complicated than what we see in the standard herb book. 

Aside from immune stimulating activity, Echinacea is also used for immune modulating effects, reducing inflammation for example. Immune modulation by Echinacea is due partly to plant alkylamides, whose effects are mediated in part via cannabinoid receptors on immune cells (5). Alkylamides, for example, suppress the production of pro-inflammatory molecules such as Tumor Necrosis Factor ?  (TNF-?) by macrophages. As mentioned, it’s possible to grow plants lacking endophytes.  Endophyte-free Echinacea plants had lower levels of alkylamides compared to plants innoculated with bacterial (6) or fungal (7) endophytes isolated from other Echinacea plants. In other words, the presence of certain endophytes increases alkylamide levels in Echinacea; yet another way endophytes significantly influence this commonly used and much valued medicine. 

Ashwagandha 

OK, so Echinacea is more that what it seems at first glance.  How about another extensively used medicinal plant (and one of my favorites), Ashwagandha? 

As you can probably guess, given the topic of this class and article, endophytes are at it again…  

Baby Ashwagandha & Milo

Withanolides are steroidal lactones and are the best studied constituents of Ashwagandha. Withanolides have a broad range of activities:  Anti-inflammatory, anti-oxidant, anti-tumor, radioprotective, anti-bacterial, immunomodulating, liver protective, anti-rheumatic, anti-anxiety, etc etc etc….(8).  Both bacterial and fungal endophytes significantly increase withanolide production in Ashwagandha root and other parts of the plant (9, 10) by activating plant genes involved in withanolide synthesis (9). For those who care, withaferin A, withanolide A, withanolide B and 12-deoxy-withstramonolide all went up. Withafarin A was not detectable in Ashwagandha roots lacking endophytes, but was found in the roots in significant amounts in plants innoculated with certain endophytes (9). For other microbiology geeks out there, some of the responsible bugs found in roots, leaf and stem included the bacteria Bacillus, Staphylococcus and Pseudomonas and the fungi Aspergillus and Penicillum (9). 

What is really cool is that some endophytes, like the fungus Taleromyces pinophilus, can make withanolides on their own, and production by fungal isolates was more robust than by the plant itself (11)!  How is it that a fungus can make the same “plant medicine” as the plant itself?  Not clear. Possibly, it’s an example of “horizontal” gene transfer between species. (As opposed to “vertical” gene transfer, which is inheritance from parent(s) to offspring.) Somehow the fungus seems to have picked up Ashwagandha genes.

Ginkgo

I’ve been on Ginkgo since getting kicked in the head by a spazz of a training partner. Between that and dementia on both sides of the family, taking Ginkgo seems like a good idea. Ginkgo leaf extracts improve cognitive function via better cerebral blood flow as well as by acting as a brain anti-oxidant:  Rust-o-leum for the noodle, as it were.  Ginkgo also improves circulation to other parts of the body, including the reproductive system, and has protective effects on the cardiovascular system.  Ginkgo may even have some cancer preventative effects, showing activity in cell culture and animal models (12). Some well known Ginkgo metabolites contributing to its medicinal effects include terpenoids such as the creatively named bilobalide and ginkgolides, along with multiple flavonoids.  

Ginkgo’s resident critters represent the various ways that endophytes influence the secondary metabolites found in plants. For example, the fungal entophyte Pestalotiopsis uvicola can make bilobalide independent of the plant (13). Another fungal endophyte, Fusarium oxysporum, can synthesize ginkgolide B (14), while Aspergillus species synthesize Ginkgo flavonoids (15).   Other fungal endophytes can increase Ginkgo’s own production of flavonoids (16). And then there’s a fungal endophyte, Chaetomium globosum, that produces anti-tumor metabolites called chaetoglobosins that the Ginkgo itself doesn’t make (17). 

I think this is interesting in and of itself simply from a nerd’s point of view. But, just think…if fungi can produce these things in culture, maybe nutraceutical companies will start leaving the plants alone and work with microbial cultures to produce medicinal constituents. Less pressure on the plant.  I know, you’re grumbling right now that plant medicine is more than just a few isolated chemicals.  I agree fully.  But, hey, if a company is obsessed with the “active principles”, let ‘em produce them in a dish instead of razing the countryside. 

Tulsi

On to Tulsi (aka Holy Basil), the “Queen of Herbs” in Ayurveda.  Among other uses, Tulsi has traditionally been turned to for diabetes. Tulsi leaf extracts reduce blood sugar in part by inhibiting alpha – amylase, a key enzyme in breaking down starch and glycogen into simple sugars. I wonder how much of this activity is due to the plant itself versus resident endophytes, given that metabolites produced by the fungal endophytes Alternaria, Colletotrichum, Diaporthe and Trichoderma isolated from Tulsi leaf and twig strongly inhibited not only alpha – amylase but also alpha – glucosidase (18), which breaks starch and disaccharides down into glucose. In addition, the endophyte metabolites inhibited aldose reductase (18), an enzyme in glucose metabolism that contributes to diabetic complications in the eyes, kidneys and elsewhere. 

Multiple studies show the potential for Tulsi in cancer prevention (19), and the fungal Alternaria, Phoma and Exserohilum may contribute by synthesizing metabolites able to kill cancer cells (20).  At least, cells in a dish, keeping in mind that this doesn’t always translate to efficacy in an actual person. 

Tulsi is useful for dealing with infections of various ilk, being broadly active against pathogenic microbes. It’s possible that its endophytic hitchhikers play some role in this. For example, the fungal endophytes mentioned above also have activity against several pathogenic bacteria (20), and other endophytes from Tulsi can stimulate the activity of neutrophils (21), which fight infection as part of the innate immune response.

Tulsi has been used also for its organ-protective effects:  Liver, kidneys, brain and such. Crude fractions of the fungus Paecilomyces isolated from Tulsi protected the liver from oxidative damage in experimental models (22), so maybe endophytes contribute to Tulsi’s protective effects as well. To be clear, how involved endophytes are in these various activities of Tulsi — enzyme-inhibition, cancer prevention, resolving infection, organ health — presumably depends on the significance of the impact on the secondary metabolites present within the plant. So, it’s simply not yet clear. 

One more note on Tulsi. It has a very characteristic scent.  Whether it’s the Krishna, Rana or Vana variety, it’s still identifiable as Tulsi even though the essential oil chemistry differs somewhat among these varieties.  Tulsi’s endophytes may influence the characteristics and strength of the scent. This goes beyond a pleasant scent, as plant volatile oils are often a significant part of the medicine.  The fungal endophyte Macrophomina phaseolina is abundant in Tulsi leaf and produces the volatile oil 2H- pyran-2-one, 5,6-dihydro-6-pentyl (24).  (Say that 5 times fast.)  That mouthful is otherwise known as 10C-Massia lactone, which has a creamy, sweet, fruity scent (25). Bacterial endophytes, including Bacillus subtilis, can enhance both plant volatile oil production as well as plant size (23).  Perhaps something to be taken advantage of in large scale essential oil production…fewer plants for the same amount of oil.

Red Sage

Red Sage root (Danshen) is valued in modern Chinese Medicine for cardiovascular benefits.  Red Sage is immune modulating, anti-oxidant (really, is there a medicinal plant that isn’t?), anti-tumor, anti-inflammatory, anti-microbial…the usual collection of “antis”.  Metabolites characterized so far include flavonoids, terpenoids, salvianolic acids and other phenolics. The most well characterized are diterpenoids known as tanshinones, which contribute to all of the activities noted above (26). 

Chaetomium globosum, a fungal isolate from Red Sage root (and also of Ginkgo fame!), can significantly increase tanshinone production in the root (27). In the absence of this endophyte, tanshinone levels were quite low (27). Levels of salvainolics acid were also bumped up and root and overall plant size were increased as well (27).   Salvainolic acids have multiple ways of protecting the heart and vasculature from damage. 

Other endophytes from Red Sage synthesize flavonoids, saponins, steroids, tannins, alkaloids, terpenoids and phenolics, including salvionolic acid C, that the plant also makes (28, 30); another case of the bugs making some of the same medicines the plant makes. Again, an increase in plant size was seen in response to certain endophytes (29), and some endophytes could even synthesize metabolites — cardiac glycosides and anthroquinones — not otherwise found in the plant (28). Cardiac glycosides have obvious relevance to the cardiovascular system, and some anthroquinones may impact blood vessel contractility. Interesting, given the use of Red Sage for the cardiovascular system.  

Guduchi (Tinospora cordifolia)

Based on the most of the plants in this article, you may be guessing that a lot of the research on endophytes in medicinal plants is being carried out in Asia, and you’d be correct. Along those lines, there is a growing pile of info on how endophytes impact Guduchi, another valued medicine from the Ayurvedic tradition. Yet another multifaceted herbal medicine, Guduchi is commonly used, among other things, for urinary issues ranging from gravel and stones to bladder weakness.  TMI warning. When on Guduchi, even for less than a week, I only had to get up to pee once at night instead of the usual 2-3 times. (Why am I not still on it ????)  

Guduchi is also used for gout, and it’s been a component in my formulas the few times I’ve worked with this.  Gout is the, at times excruciating, accumulation of uric acid in joints, particularly in but not limited to the big toe. Gout sucks. Xanathine oxidase is an enzyme involved in uric acid production. Several endophytes isolated from Guduchi make inhibitors of xanathine oxidase, perhaps contributing to Guduchi’s benefits in gout (31).  So, again, a case where the endophytes may be the source of some of the activity of our herbal medicines.

Guduchi extracts have shown various anti-tumor activities in preclinical trials (ie. cell culture and animal models).  A fungal endophyte isolated from Guduchi, Fusarium culmorum, produces Taxol (32), one of the most widely used chemotherapy drugs. Another endophyte, Cladosporium velox, produces a bunch of polyphenols — gallic acid, catechin, chlorogenic acid, epicatechin, caffiec acid, coumaric acid, ellagic acid and others — with with genoprotective and anti-oxidant effects (33). Genoprotective agents reduce DNA mutations and that’s relevant to cancer because, with only a handful of exceptions, cancer has gene mutations as it’s root.  

Others…

St. John’s Wort

Not to be left out, St. John’s Wort has a fungal endophyte (Thielavia subthermophila) that makes  hypericin (34), while root-associated mycorrhial fungi can stimulate increased production of both hypericin and pseudohypericin by St. John’s Wort itself (35).

Those of you who use Milk Thistle may be familiar with silymarin, a seed extract containing over a half dozen flavinoligan compounds and used for hepatoprotective effects.  A few of these flavinoligans  — Silybin A, silybin B, and isosilybin A — can be made by a fungal endophyte, Aspergillus iizukae, that lives in the leaves of the plant (36). 

Ginsenosides are steroidal saponins in Panax Ginseng that contribute significantly to the many uses of this vastly popular plant medicine.  So far we know that two of these, ginsenoside Rb2 and ginsenoside Rc, can be made by fungal endophytes from the root (37). 

And there are many, many more examples of how our favorite medicines may be slightly more than just “herbal” medicines.  To summarize, the various outcomes of endophyte-plant interactions on secondary metabolite production include:  (i) endophytes making something that the plant doesn’t make, (ii) endophyte stimulating the plant to make something (or to make something at higher levels) and (iii) both endophyte and plant making the same metabolites.  This last one seems the most weird.  What’s the benefit to endophyte or plant when both are making the same metabolites? Who knows….

Anyway, what’s the point of all of this?  A big part of it is me geeking out on some cool microbiology.  Research into endophytes and medicinal plants has grown quite a bit over the past 10 years.  More practically, there’s the thought that adding certain endophytes to farmed medicinal plants may improve plant size and yields, cutting down on environmental impact and, hopefully, helping smaller growers make a better living.  That said, there are potential issues with sprinkling endophytes willy nilly on plants.  For instance, what’s friendly to one plant species may not be to another.  

As mentioned earlier, crude extracts are where it’s at for many herbalists, including myself. Most standardized extracts seem to be bullshit, as additional “active principles” keep being found for various plants that are marketed as standardized extracts (eg. Turmeric, St. John’s Wort, others). You may disagree. You may not. And, isolated components, such as curcumins, lack the full effect of the crude extract and have more side effects. But, I’ll mention again that I do like the idea of having alternate sources of secondary metabolites, cultured endophytes, so that nutraceutical companies are perhaps less likely to wipe out plants x, y or z to obtain such metabolites for their products.  

References

1. Haron, MH, et al (2014) Immune enhancing Echinacea bacterial endophytes. Planta Med. 80 – PP15.  https://www.thieme-connect.com/products/ejournals/abstract/10.1055/s-0034-1382710

2. Todd, DA, et al (2015) Ethanolic Echinacea purpurea extracts contain a mixture of cytokine-suppressive and cytokine-inducing compounds, including some that originate from endophytic bacteria. PLOS One. 10(5): e0124276.  https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124276

3. Pugh, ND, et al (2008) The majority of in vitro macrophage activation exhibited by extracts of some immune enhancing botanicals is due to bacterial lipoproteins and lipopolysaccharides. Int Immunopharmacol.  8(7):1023-32.   https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2467439/

4. Tamta, H, et al (2008) Varability in in vitro macrophage activation by commercially diverse bulk Echinacea plant material is due predominantly to bacterial lipoproteins and lipopolysaccharides. J Agric Food Chem. 56(22):10552-10556.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2670557/pdf/nihms91114.pdf

5. Raduner, S, et al (2006) Aklylamides from Echinacea are a new class of cannabinomimetics: CAnnabinoid type 2 receptor-dependent and -independent immunomodulatory effects.  J Biol Chem. 281:14192-06. http://www.jbc.org/content/281/20/14192.full.html

6. Maggini, V, et al (2017) Plant-endophytes interaction influences the secondary metabolism in Echinacea purpurea (L.) Moench: an in vitro model. Scientific Rep. 7:16924. https://www.nature.com/articles/s41598-017-17110-w

7. Gualandi, RJ Jr. (2010) Fungal endophytes enhance growth and production of natural products in Echinacea purpurea (Moench.). ” Master’s Thesis, University of Tennessee. https://trace.tennessee.edu/utk_gradthes/713/

8. Budhiraja, RD, et al (2000) Biological activity of withanolides. J Sci Indust Res. 59:904-11. buhttp://nopr.niscair.res.in/bitstream/123456789/26628/1/JSIR%2059(11)%20904-911.pdf

9. Pandey, SS, et al (2018) Endophytes of Withania somnifera modulate in planta content and the site of withanolide biosynthesis. Scientific Reports.  8:5450. https://www.researchgate.net/publication/324173010_Endophytes_of_Withania_somnifera_modulate_in_planta_content_and_the_site_of_withanolide_biosynthesis

10. Mishra, A et al (2018)  Bacterial endophytes modulates the withanolide biosynthetic pathway and physiological performance in Withania somnifera under biotic stress. Microbiol Res. Jul – 212-213:17-28. https://www.ncbi.nlm.nih.gov/pubmed/29853165

11. Sathiyabama, M & R Parthasarathy (2018) Withanolide production by fungal endophyte isolated from Withania somnifera. Nat Prod Res. 32(13):1573-77.  https://www.ncbi.nlm.nih.gov/pubmed/29034745

12. DeFeudis, FV, et al (2003) Ginkgo biloba extracts and cancer: A research area in it’s infancy. Fundam Clin Pharmacol. 17(4):45-17. https://www.ncbi.nlm.nih.gov/pubmed/12914542

13. Qian, Y-X, et al (2016) A bilobalide-producing endophytic fungus, Pestalotiopsis uvicola from medicinal plant Ginkgo biloba. Curr Microbiol. 73(2):280-6.  https://link.springer.com/article/10.1007/s00284-016-1060-6

14. Cui, Y, et al (2012) Ginkgolide B produced endophytic fungus (Fusarium oxysporum) isolated from Ginkgo biloba. Fitoterapia 83(5):913-20. https://www.sciencedirect.com/science/article/pii/S0367326X12001165

15. Qiu, M, et al (2010) Isolation and identification of two flavonoid-producing endophytic fungi from Ginkgo biloba L. Annal Microbiol. 60(1):143-150.  https://www.researchgate.net/publication/225651927_Isolation_and_identification_of_two_flavonoid-producing_endophytic_fungi_from_Ginkgo_biloba_L

16. Hao, G, et al (2010) Fungal endophytes-induced abscisic acid is required for flavonoid accumulation in suspension cells of Ginkgo biloba. Biotechnol Let. 32(2): 305–314.   https://link.springer.com/article/10.1007/s10529-009-0139-6

17. Li, H, et al (2014) Chaetoglobosins from Chaetomium globosum, an endophytic fungus in Ginkgo biloba, and their phytotoxic and cytotoxic activities.  J Agric Food Chem. 62(17):3734-41. https://www.researchgate.net/publication/261441769_Chaetoglobosins_from_Chaetomium_globosum_an_Endophytic_Fungus_in_Ginkgo_biloba_and_Their_Phytotoxic_and_Cytotoxic_Activities

18. Pavritha, N, et al (2014) In-vitro Studies on ?-Amylase, ?-Glucosidase and Aldose Reductase Inhibitors found in Endophytic Fungi Isolated from Ocimum sanctum.  Curr Enz Inhibition. 10:129-36. https://www.researchgate.net/publication/269694533_In-vitro_Studies_on_a-Amylase_a-Glucosidase_and_Aldose_Reductase_Inhibi-_tors_found_in_Endophytic_Fungi_Isolated_from_Ocimum_sanctum

19. Baliga, MS (2013) Ocimum sanctum L (Holy Basil or Tulsi) and its phytochemicals in the prevention and treatment of cancer. Nutr Cancer. 65 Suppl 1:26-35.   https://www.ncbi.nlm.nih.gov/pubmed/23682780

20. Bhagat, J, et al (2010) Molecular and functional characterization of endophytic fungi from traditional medicinal plants. World J Microbiol Biotechnol. 28(3):963-71.https://www.ncbi.nlm.nih.gov/pubmed/22805817

21. Madagundi, S, et al (2013) Free radical scavenging and in vitro immunomodulatory activites of endophytic fungi of Ocimum sanctum Linn. Farmacia. 61(2):330. https://www.researchgate.net/publication/261418046_FREE_RADICAL_SCAVENGING_AND_IN_VITRO_IMMUNOMODULATORY_ACTIVITIES_OF_ENDOPHYTIC_FUNGI_OF_OCIMUM_SANCTUM_LINN

22. Shukla, ST, et al (2012) Hepatoprotective and antioxidant activities of crude fractions of endophytic fungi of Ocimum sanctum Linn. Oriental Pharm Exp Med. 12(2):81-91. https://www.researchgate.net/publication/257805712_Hepatoprotective_and_antioxidant_activities_of_crude_fractions_of_endophytic_fungi_of_Ocimum_sanctum_Linn_in_rats

23. Tiwari, R (2010) Endophytic Bacteria from Ocimum sanctum and Their Yield Enhancing Capabilities. Curr Microbiol. 60(3):167-71. https://www.researchgate.net/publication/26892447_Endophytic_Bacteria_from_Ocimum_sanctum_and_Their_Yield_Enhancing_Capabilities

24. Chowdhary K, Kaushik N (2015) Fungal Endophyte Diversity and Bioactivity in the Indian Medicinal Plant Ocimum sanctum Linn. PLoS ONE 10(11): e0141444. 90  https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141444

25. http://www.thegoodscentscompany.com

26. Wang, B-Q (2010) Salvia miltiorrhiza: Chemical and pharmacological review of a medicinal plant. J Med Plant Res. 4(25):2813-20.http://www.academicjournals.org/app/webroot/article/article1380714402_Wang.pdf

27. Zhai, X, et al (2018) Endophyte Chaetomium globosumD38 Promotes Bioactive Constituents Accumulation and Root Production in Salvia miltiorrhiza. Front Microbiol.

8:2694. https://www.frontiersin.org/articles/10.3389/fmicb.2017.02694/full

28. Li, Y, et al (2015) The endophytic fungi of Salvia miltiorrhiza Bge.f. alba are a potential source of natural antioxidants. Bot Stud. 56: 5  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430307/

29. Zhou, LS, et al (2018) The Plant Growth-Promoting Fungus (PGPF) Alternaria sp. A13 Markedly Enhances Salvia miltiorrhiza Root Growth and Active Ingredient Accumulation under Greenhouse and Field Conditions. Int J Mol Sci. 19(1):270. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796216/

30.  Li, X, et al (2016) Phoma glomerata D14: An Endophytic Fungus from Salvia miltiorrhiza That Produces Salvianolic Acid C. Curr Microbiol. 73(1):31-7. https://www.researchgate.net/publication/298740061_Phoma_glomerata_D14_An_Endophytic_Fungus_from_Salvia_miltiorrhiza_That_Produces_Salvianolic_Acid_C

31. Kapoor, N & S Saxena. (2018) Endophytic fungi of Tinospora cordifolia with anti-gout properties. 3 Biotech. 8(6):264.  https://www.researchgate.net/publication/325254828_Endophytic_fungi_of_Tinospora_cordifolia_with_anti-gout_properties

32. Visalakchi, S, et al (2010) Taxol producing endophytic fungus Fusarium culmorum SVJM072 from medicinal plant of Tinospora cordifolia – a first report. J Biotechnol. 150:425-425. https://www.researchgate.net/publication/241082625_Taxol_producing_endophytic_fungus_Fusarium_culmorum_SVJM072_from_medicinal_plant_of_Tinospora_cordifolia_-_a_first_report

33. Singh, B, et al (2016)  Antioxidant and in vivo genoprotective effects of phenolic compounds identified from an endophytic Cladosporium velox and their relationship with its host plant Tinospora cordifolia.  J Ethnopharmacol. 194:450-6. https://www.researchgate.net/publication/308993108_Antioxidant_and_in_vivo_genoprotective_effects_of_phenolic_compounds_identified_from_an_endophytic_Cladosporium_velox_and_their_relationship_with_its_host_plant_Tinospora_cordifolia

34. Kusari, S, et al (2008) J Nat Prod. 71(2):159-162.  An endophytic fungus from Hypericum perforatum that produces hypericin. https://www.ncbi.nlm.nih.gov/pubmed/18220354

35. Zubek, S, et al (2012) Hypericin and pseudohypericin concentrations of a valuable medicinal plant Hypericum perforatum L. are enhanced by arbuscular mycorrhizal fungi. Mycorrhiza. 22(2):149–156. https://www.researchgate.net/publication/51178213_Hypericin_and_pseudohypericin_concentrations_of_a_valuable_medicinal_plant_Hypericum_perforatum_L_are_enhanced_by_arbuscular_mycorrhizal_fungi

36.El-Elimat, T, et al (2014) Flavonolignans from Aspergillus iizukae, a fungal endophyte of milk thistle (Silybum marianum). J Nat Prod 77(2):193-9. https://libres.uncg.edu/ir/uncg/f/H_Raja_Flavonolignans_2014.pdf

37. Wu, H, et al (2013) Diversity of endophytic fungi from roots of Panax ginseng and their saponin yield capacities 

 SpringerPlus. 2:107. https://www.researchgate.net/publication/236095071_Diversity_of_endophytic_fungi_from_roots_of_Panax_ginseng_and_their_saponin_yield_capacities

Defeudis, FV (2002) Bilobalide and neuroprotection. Pharmacol. Res. 46(6):565-8. https://www.sciencedirect.com/science/article/abs/pii/S1043661802002335.

Endophytes: Think you’re making HERBAL medicine? Think again….

Originally published in Plant Healer Quarterly

“Big fleas have little fleas upon their backs to bite ‘em.  

And little fleas have lesser fleas, and so, ad infinitum.  

And the great fleas, themselves, have greater fleas to go on, 

While these again have greater still, and greater still, and so on…”  

– Jonathan Swift 

Many of us think of ourselves and other beings as an “individuals”…Jane, Joe, Rex the dog, Tiger the cat.  In reality, life is more similar to a fractal, or perhaps a set of Russian nesting dolls.  We’re each a hodge podge of many smaller individuals; a collection of our cells as well as fungi and bacteria that do more than just tag along…they influence how we are. Don’t even get me started on the the viruses that infect our resident bacteria and fungi and influence how they are.  Even “our own” cells contain viral genetic sequences integral to our DNA. So microbial tag alongs may even influence who we are.  Not to forget the other end of the spectrum, in which we’re but one component of the larger organism of the planet.  “Individuals”, indeed. 

Plants are no exception to this nesting doll reality. When we make plant extracts, we are in fact making plant, fungal and bacterial extracts. Medicinals such as Chamomile, Mints, Saint John’s Wort, Skullcap, Rosemary, Geranium, Fennel, Artemesia and many others have been studied for their fungal and bacterial tag-alongs, called “endophytes” (1, 2), meaning “inside plants”.  In fact, all plant species tested to date contain endophytes, critters who hang around inside living tissue without causing disease. Wash the plant all you want before extracting and you don’t get rid of them. Endophytes exist either inside of or squeezed in between the plant cells. And, really, you may not want to get rid of them.  

“So what?”, you may ask. Well, endophytes may be a key determinant of the quality of our plant medicine.  As more and more research is pointing to just how much our health and emotional state are impacted by the health of our microbiome, it’s becoming clear that the growth, stress resistance and chemical make up of plants is dependent upon their resident microbiome, the endophytes. And, well, endophytes are pretty cool.  Have you heard of hypericin?  An endophyte can make it.  Diosgenin? Ditto.  Artemisin?  Yup. Taxol? You get the idea…    More on “plant” medicine momentarily.  First, let’s get in to the how, what and why of endophytes. 

Endophytes, you say?

If a plant geek, you’ve heard of mycorrhizal microbes that grow associated with plant roots. These bugs facilitate water and nutrient uptake by the roots and mediate plant-to-plant communication as well. Those that actually penetrate into the roots are endophytes. But endophytes are also found inside of seeds, leaves, stems, flowers, fruit, buds and bark (3). 

Most endophytes identified so far are filamentous fungi though many are bacteria (4), and they’re tough little bastards to study.  Most of the research looking at endophytes are folks interested in “drugifying” the metabolites that they make.  To study them scientists have to surface sterilize a plant then grind it up to release the endophytes, which they’ll attempt to culture on various growth media. But not all endophytes are willing to cooperate with this arrangement and refuse to be cultured.  Just how many different endophytes are out there?  One study alone cultivated 181 bacterial endophytes from 13 medicinal herb species (1). This, of course, doesn’t include those that can’t be grown in culture and the study didn’t look for fungal endophytes. Thus, the answer to how many endophytes are out there is “a shitload”.  

So, what do plants get out of this intimate arrangement?  In some cases, the endophyte grants the plant increased resistance to parasites or to grazing insects and animals.  Or a better likelihood of surviving changing environmental conditions. Or more robust growth.  And the endophyte?  Many survive in the soil for a long time without a plant home. But, when inside the plant, the endophyte gets necessary nutrients or completing its life cycle (3).  Though this relationship is not all roses; sometimes the relationship is antagonistic (Married with Children?) or parasitic (The Hunger?) rather than mutualistic (3).

When and how did endophytes get there initially???  Who the hell knows…  Have they been tagging along since the beginning?  Or, did plants become colonized somewhere down the line?  Plants are thought to have first set foot (er, root) on land by about 700 million years ago.  Fossil evidence points to a plant-endophyte relationship being in place by about 400 million years ago (5). So it’s certainly not a new partnership. 

As to the how, it’s known that some endophytes are transmitted vertically, meaning that they’re passed from mama to baby plants via seed. These are thought to be “obligate” endophytes that can’t exist outside of the plant (3).  Other endophytes are transmitted horizontally, meaning that they’re spread from plant to plant by endophyte spores (6). These are thought to be “facultative” endophytes, capable of hanging out in some form elsewhere but living inside of the plant for a good chunk of their lifecycle (3). So maybe at some point way back when, a spore made it’s way into a plant. After all, fungi were already hanging around on land long before plants showed up. Or, given that some plants need their endophytes in order to grow to maturity from seed (4) or to survive in a stressful environment (7), maybe endophytes were there from the very beginning and were pivotal to successful plant evolution. 

Back to medicinal plants

The existence of endophytes has been known for over a hundred years (4), yet I can’t claim to have though about them and their contribution to the medicine sitting in jars on my shelf until recently.  Plants obviously provide great medicine. Herbal medicine works because many of the secondary metabolites that aid the plants also benefit us. What’s become clear is that endophytes also make secondary metabolites, many that we typically associate with plants.

Endophytes may be influencing our plant medicine in multiple ways.  Clearly there are more plant chemicals by sheer mass than endophyte chemicals in that jar of macerating Peppermint in the cabinet.  But it’s evident that endophytes have an impact on that medicine. For example, there may be chemicals in those jars that likely wouldn’t be there if not for endophytes (8). In some cases, this is because the endophytes are synthesizing stuff that the plant doesn’t make itself. Alternatively, the endophyte may be stimulating the plant to make something it wouldn’t without the endophyte’s influence; resveratrol in Doug Fir is an example (4, 8).  Endophytes may also influence levels of metabolites the plant already makes on its own.  Echinacea’s immune modulating alkamides are an example of this (9).

Echinacea purpurea

Sometimes both the plant and the endophyte produce the same metabolite(s). In this case, the plant and endophyte may be sharing genes via gene transfer from one organism to the other, or they may have co-evolved the ability to make a particular metabolite, as seems to be the case in some Artemesia species (2). 

It’s theoretically possible that endophytes may be responsible for the primary medicinal actions of a plant in some cases. Either by directly producing strong medicinal metabolites that you don’t need a whole lot of for effects, or by influencing the plant’s production of medicinal stuff.  Endophytes themselves make a veritable cornucopia (yes, I went there) of medicinal compounds. There’s a handy table included here that was compiled for you tabley types. It lists many of the categories of secondary metabolites that endophytes produce, along with some specific examples. Anyone familiar with plant chemistry will immediately recognize that endophytes make a whole pile of metabolites that we typically think of as plant medicine.  

As mentioned, the majority of the research being carried out on endophytes is towards the discovery of novel new drugs. The relevance of endophytes is perhaps different for us herbalists…more on this monetarily. It’s known that fungal endophytes produce a larger array of secondary metabolites than bacterial do.  And, endophytes in desert and tropical plants make a wider variety of compounds than endophytes in temperate climes (4).  In any case, it’s interesting for us medicine making herbalists to know that the origin of our plant medicine is more complex than it seems at first glance.  

At this point, there are more questions than answers. For example…

  • How much is the quality of our herbal medicine determined by the influence of growth conditions on the plant itself versus on the plant’s endophytes?   It seems likely that the answer is that both are important.  Many of us consider role of growing location and conditions as key determinants of how good our plant medicine will be. Studies to date show that these factors also impact who is living inside our medicinal plants (10), which is likely influencing our medicine.  
  • Along those lines, how much do endophytes contribute to how good your Mugwort medicine is compared to the Mugwort grown in your second cousin’s garden on the other side of the country? If hers is a stronger dream herb, would it work to expose your soil and plants to her ground up Mugwort?   Research shows that environmental conditions do influence the endophytes present (10), so I wonder how such a transfer experiment would work if you live in Sedona, Arizona and your second cousin lives in Presque Isle, Maine.  I bet some endophytes will “take”.  
  • Is there a potential problem in trying endophyte transfers such as these?  A chance, for instance, of transmitting an unwanted infection by a disease-causing organism? One idea that is appealing in this age of environmental degradation and over-harvesting is the idea of an endophyte library being created to help propagate endangered medicinal plants (10).   This would also reduce the chance of transferring unwanted critters cross country.     

These meanderings may be a bit more technical than the level at which many of us work day to day, but they’re relevant to our medicine and are something to chew on until next time, when we delve deeper into the critter chemical factories within some of our best known and loved medicinal plants.

References & further reading

  1. Goryluk-Salmonowicz, A, et al (2016) Endophytic detection in selected European herbal plants. Pol J Micro. 65(3):369-75. http://www.pjmonline.org/endophytic-detection-in-selected-european-herbal-plants/

2. Huang, WY, et al (2007) Methods for the study of endophytic microorganisms from traditional Chinese medicine plants. Econom Bot. 61(1): 14-30. https://www.jstor.org/stable/4257167?read-now=1&loggedin=true&seq=1#page_scan_tab_contents

3. Gouda, S, et al (2016) Endophytes: A treasure house of bioactive compounds of medicinal importance. Frontiers in Microbiology. 7:1583. https://www.frontiersin.org/articles/10.3389/fmicb.2016.01538/full  REVIEW

4. Owen, NL & N Hundley (2004) Endophytes — The chemical synthesizers inside plants. Science Progress. 87(2):79-99. https://www.jstor.org/stable/43423175?seq=1#page_scan_tab_contents   REVIEW

5. Krings, M, et al (2007) Fungal endophytes in a 400-million-yr-old land plant: infection pathways, spatial distribution, and host responses. New Phytol. 174(3):648-57. https://www.researchgate.net/publication/6380784_Krings_M_Taylor_TN_Hass_H_Kerp_H_Dotzler_N_Hermsen_EJ_Fungal_endophytes_in_a_400-million-yr-old_land_plant_infection_pathways_spatial_distribution_and_host_responses_New_Phytol_174_648-657

6. Kaul, S, et al (2012)  Endophytic fungi from medicinal plants: a treasure hunt for bioactive molecules.  Phytochem Rev. 11(4):487-505. https://www.academia.edu/17123332/Endophytic_fungi_from_medicinal_plants_a_treasure_hunt_for_bioactive_metabolites   REVIEW

7. Rodriguez, R & R Redman (2008)  More than 400 million years of evolution and some plants still can’t make it on their own: plant stress tolerance via fungal symbiosis.  J Exp Biol. 59(5):1109-14. https://academic.oup.com/jxb/article/59/5/1109/538568

8. Huang, L-H, et al (2018) Endophytic fungi specifically introduce novel metabolites into grape flesh cells in vitro. PLOS One. 13(5): e0196996.   https://doi.org/10.1371/journal.pone.0196996

9. Maggini, V, et al (2017) Plant-endophytes interaction influences the secondary metabolism in Echinacea purpurea (L.) Moench: an in vitro model. Sci Rep. 7: 16924. https://www.nature.com/articles/s41598-017-17110-w

10. Jia, M, et al (2016) A friendly relationship between endophytic fungi and medicinal plants: A systemic review. Front. Microbiol. 7:906. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899461/   REVIEW

11. Yu, H, et al (2010) Recent developments and future prospects of antimicrobial metabolites produced by endophytes. Microbiol. Res. 165(6):437-449. https://www.sciencedirect.com/science/article/pii/S0944501309001128  REVIEW

12. Gunatilaka, AAL (2012) Natural products from plant-associated microorganisms: Distribution, structural diversity, bioactivity and implications of their occurrence. J Nat Prod. 69(3):509-26.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362121/  REVIEW

13  Golinska, P, et al (2015) Endophytic actinobacteria of medicinal plants: diversity and bioactivity.    Antonie van Leeuwenhoek. 108:267–289.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491368/  REVIEW

14. Venieraki, A, et al (2017) Endophytic fungi residing in medicinal plants have the ability to produce the same or similar pharmacologically active secondary metabolites as their hosts. Hellenic Plant Prot J. 10:51-66.  https://www.researchgate.net/publication/318656074_Endophytic_fungi_residing_in_medicinal_plants_have_the_ability_to_produce_the_same_or_similar_pharmacologically_active_secondary_metabolites_as_their_hosts REVIEW

15. Kual, S, et al (2013) Prospecting endophytic fungal assemblage of Digitalis lanata Ehrh. (foxglove) as a novel source of digoxin: a cardiac glycoside. 3 Biotech. 3(4): 335-40. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3723867/

16. Kuar, A, et al (2017) Secondary metabolites from fungal endophytes of Echinacea purpurea suppress cytokine secretion by macrophage-type cells. Nat Prod Commun. 11(8):1143-6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414731/

17. Nicoletti R & A Fiorentino (2015) Plant bioactive metabolites and drugs produced by endophytic fungi of spermatophyta. Agriculture. 5:918-970.  http://www.mdpi.com/2077-0472/5/4/918/htm

18. Zin Z, et al (2017) Antimicrobial activity of saponins produced by two novel endophytic fungi from Panax notoginseng. 31(22):2700-03.  https://www.tandfonline.com/doi/abs/10.1080/14786419.2017.1292265

19. Lu, H et al (2000) New bioactive metabolites produced by Colletotrichum sp., an endophytic fungus in Artemesia annua. Plant Sci. 151(1):67-73. http://www.paper.edu.cn/scholar/showpdf/NUz2MN2INTz0kxeQh

Mushromatherapy – Part 1

Artist’s Conk / Ganoderma applanatum

Any of you who’ve stuck your nose up to a mushroom know that they are aromatic.  Even if they just smell like “mushroom”, which, by the way, is largely due to the chemical octanol and its relatives. Some mushrooms smell wonderful (fresh Oyster Mushrooms in the forest); others, not so much (Stinkhorn!).

Mushroom volatile chemicals have been less sussed out than those of plants.  “Volatile” simply means that a molecule evaporates readily at ambient temperature.  Anyway, there are over 100,000 described fungal species. Aromatics have been assessed in maybe 100 of these (1), and, not all of these are fungi of the “mushroom” persuasion. Most of the info on mushroom aroma comes from work done by ecologists studying interactions among species and by folks in the food or perfume industry.  

Mushroom volatiles fall into the chemical categories that you aromatherapists will recognize from plants. (Nature doesn’t often reinvent the wheel.) These include hydrocarbons, terpenes, ketones, acids, aldehydes, alcohols, esters, hetero-cycles, nitrogen-containing compounds thiols (sulfur-containing…stinky!) and aromatics (confusingly, referring to chemical structure rather than scent)(1). We’re inhaling these in while walking in the woods, and also when stuck inside a musty, old building.  

And, not all mushroom odors are actually made by the mushroom.  Some, instead, come from other fungi or even bacteria living on or in the shroom. Truffles are prized for their distinctive scent, but the bacteria that hitchhike along with Truffles should get a big part of the credit (1).  Others mushroom aromatics come from the substrate that the mushroom is “eating” (1).

Why do mushrooms have an aroma?

For the same reasons plants do:  Growth, development, reproduction, competition and protection. Some mushroom aromatics may regulate if and when a mushroom sporulates. Others may protect the fruiting body from being eaten, or may lure in “pollinators” to spread the mushroom spores.  Many mushroom volatile compounds are anti-microbial, protecting from infection by bacteria, protists and other fungi. 

Mushrooms also communicate with other fungi, along with bacteria and plants, using their volatile messengers. The volatiles may be telling these other organisms to piss off or to come hither. Or, might tell them something along the lines of “Holy shit, some asshole is digging up the forest…protect yourselves!”.  Mushroom aromatics may stimulate plant growth, which is useful if that plant is the mushroom’s host. Other mushroom aromatics may inhibit plant growth, which may be useful if that particular plant competes with the mushroom’s host plant. 

Volatile chemicals produced by fungi (and bacteria) in the soil can carry out communication long distance by traveling through air pockets in the soil. Tiny amounts of these so-called “semiochemicals” are enough to deliver the message. 

Do mushroom aromas influence us?

Oh yes.  Ever heard of “forest bathing”?  Known as Shinrin-yoku in Japan, forest bathing is an therapy that entails getting thine ass out into nature. The purpose is to appreciate the beauty while doing a little forest aromatherapy. Both of these result in multiple health benefits. Tree aromatics get all the attention, but fungal aromatics are there, too.  That rich, earthy, fungusy scent you’ve probably smelled in the woods comes, indeed, from fungi. One of the most prevalent mushroom volatiles, 1-octen-3-ol (aka. the aforementioned octenol), is sedating and may contribute to the stress relief of walking in the woods.

Red Belted Conk (Fomitopsis schrenkii)

Then, of course, there are the wonderful scents when cooking after a good forage.  Though heating mushrooms alters many of their aromatic chemicals, they still smell damned good!   Boletes roasting in the oven.  Chanterelles sauteeing on the stove.  Truffle oil…  (OK, I’ve never and never will find a Truffle.)   

Mushroom aromatics are being investigated as anti-bacterial agents, given the distressing and rapid rise in multidrug resistance in pathogenic bacteria. They also have application in food safety as a way to avoid synthetic food preservatives.  Other mushroom aromatics support the respiratory system, and some have anti-cancer activity (in a dish, at least…).  

Not all aromatics produce by fungi are good for us.  An example is the  phenomenon of “sick building syndrome” —  a collection of symptoms such as skin irritation, respiratory distress, fatigue and other fun ones — may be linked in part to fungal volatiles (1). 

——————————————————

Mushroom Perfume?!

Why not?  The scent of mushrooms goes well beyond “mushroomy”.  If you spend time in nature, you may have encountered Oyster or Horse Mushrooms with their lovely almondy/aniseed scent. (I’ve yet to find Oysters in the grocery store or from a kit with this scent…growing conditions are important.) You Pacific Northwesterners may know the spicy, pungent scent of Matsutake, which has been described by mushroom expert, David Arora, as a “provocative compromise between red hots and dirty socks”. Chanterelle aficionados are familiar with the mushroom’s apricot-like scent, whether stumbling across them in the Doug Fir forests of the PNW or finding them them half hidden under rocks in the Southern Rockies. 

The scent of mushrooms varies wildly and many have distinctively un-mushroomy scents. For example:  Grapefruit and anise (Honey Mushroom); jasmine, rose and fruit (Inocybe species); orange blossom (Flowery Blewit); burnt toffee (Trembling Merulius); pineapple, apples and coffee (Birch Polypore); fruity, banana (Bleeding Broadleaf Crust); and, not to be left out, fish and tallow (Turkey Tails)(2).  

Many volatile chemicals from fungi are used in perfumery. It’s less common for mushroom distillations or other mushroom extracts to be used in perfumery, though some are out there.  You’ll get to smell some mushroom hydrosols and blends in this class. In the  mean time, check out natural perfumer Mandy Aftelier’s Cepes (Boletus edulis) and Tuberose. There’s even a (patented) Bolete essential oil (3).  Bolete is a base note (LINK: https://basmati.com/2017/11/09/essential-oil-essentials-making-natural-perfume-simply): A heavy note that grounds a blend and gives it some heft. The larger molecular size of base notes slows the evaporation of smaller scent molecules in a perfume blend, making the perfume last longer.  The middle notes, of intermediate size, round out a perfume blend. 

Rocky Mountain Red (Boletus rubriceps) – our local version of King Bolete

OK, back to mushrooms. Quite a few shrooms are rich in top notes, those small scent molecules that evaporate the quickest. Take the most ubiquitous mushroom aromatic, octenol. I assumed that a mushroomy aromatic such as this would be a base note.  Nope. Small molecule. Top note. 

Compared to aromatic plants such as Rosemary, Lavender, Pine and such, mushrooms don’t produce nearly the level of volatile oils. I’ve not been able to isolate mushroom essential oils either with my janky stove-top pot-with-upside-down-lid setup or with my microwave distiller. So I’ve been making hydrosols and infused oils.  We had a non-existent mushroom season in 2018 (after I proposed this class, of course), so I didn’t get to play as many wild mushrooms as I wanted. Though there were conks, of course. You’ll get to smell some mushroom (mostly conk) hydrosols and blends in this class.

Here are some of the most prevalent mushroom aromatics

C8 Compounds

The most common mushroom aromatics are aliphatic eight-carbon (“C8”) compounds. Aliphatic means that the carbons form a chain rather than a ring stucture.  They’re linoleic acid breakdown products (1) found in essentially every mushroom tested, as far as I can tell. C8s are very prevalent in the common grocery store Button Mushroom (Agaricus bisporus), making up from 44 to over 90% of the aromatic molecules (4). This relatively “boring” mushroom actually has over 150 different scent molecules, by the way (4).

A very prevalent C8 aromatic is the aforementioned octenol, which contributes significantly to mushroom scent and flavor. In addition to mushroomy, the scent of octenol has been described as lavender-like, herbal, bright, earthy, buttery, resinous, vegetable and hay-like (2, 5). You’ll get to decide for yourself in the Mushromatherapy class in May. Why such a wide range of descriptors?  Because, molecularly-speaking, octenol has 2 different configurations that result in different scents.

Octenol has a role in mushroom metabolism and sporulation.  It’s also anti-bacterial and anti-fungal (4), protecting the fruiting body from infection by unwanted critters. The scent can act as either an insect repellent or attractant depending on the bug (1). This may serve either to protect the fruiting body from being nibbled on by insects or, instead, to lure in a 6-legged carrier to help spread the mushroom spores afar. (Mushroom pollinators, as it were.)  Mosquitoes are one of the insects that octenol attracts. So, if you find yourself in a campground and there’s a noisy, annoying group there as well, sneak over and spray their camp site with octenol.   

Octenol was sedating in rodent studies (6).  Accordingly, there’s a post on the outstanding mushroom blog Reishi and Roses that describes how ridiculously sedating the aroma of Artist’s Conk was during distillation (7). Octenol is is one of the major aromatics of Artist’s Conk (8).

Artist’s Conk

Aside from mushrooms, octenol is also found in basil, spearmint, thyme, elder flowers, dill, hyssop, lavender, marjoram, oregano, rosemary, sage, lemon balm and others. Lots of mints on the list. I wonder if any of the relaxing quality of mints is related to octanol…   Pumpkins, bananas, black currants and raspberries also contain octenol (5).  And, weirdly enough, octenol’s in our breath, too.  Who knew?  

A related and even mushroomier-smelling C8 is 1-octen-3-one; and there are a slew of other C8s with scents ranging from mushroomy, buttery, nutty, fatty or metallic to floral, lavender-like, sweet, green or vegetable-like (9-10). 

Hexanal

This is the same as “1-hexanal”.

Hexanal is a major volatile component in King Boletes (12), and also lends it’s scent to those aromatic lovelies Chanterelles (13), Matsutake (14) and Oyster Mushrooms (15). Hexanal is also an aromatic in Button and Meadow Mushrooms (16).

This linoleic acid derivative and aldehyde is a yummy top note with a scent described as woody, citrusy, green, fresh, grassy, apple-like and vegetable-like (5).  Again, you’ll get to offer your own 2 cents on this one at the conference.   “Apple-like” isn’t too much of a stretch, given that hexanal’s found in apples. It’s in many other fruits and veggies, too:  Bananas, coconuts, carrots, avocados, cucumbers and a whole passel of citruses (5). Like benzaldehyde, hexanal is a popular natural flavor in the food industry and is also used to flavor rum (5).

Hexanal is also an essential oil component of herbs including parsley, oregano, bay laurel, angelica seed, clove, cayenne, oregano and spearmint.  Coffee and tobacco have it, too (5). It’s a major contributor to the green odor of leaves (17).

Hexanal inhibits plant seed germination and pollination of several plant species (18), making me wonder if some mycorrhizal mushrooms use it to inhibit competitors of their plant hosts.

Hexanal is active against nasty pathogenic bacteria such as E. coli, Salmonella and Listeria (19) and is also anti-fungal (20) and anti-viral (21). On another note, hexanal is emitted by bed bugs when they’re doing it (22). 

Hexanal is detected in the breath of folks with Multiple Sclerosis and is this is being explored as a possible detection/diagnostic test (23). Lipid peroxidation is involved in neurodegenerative diseases, and hexanal is an end product of lipid peroxidation; perhaps a reason it’s detectable in the breath of those with Multiple Sclerosis (23). 

Hexanol

This is frequently found in the literature as 1-hexanol or hexan-1-ol.  

Don’t confuse this alcohol (-ol) with the aldehyde (-al) hexanal. Hexanol is found in Oyster Mushrooms (24), Chanterelles, Truffles (1), Boletes (16) and Matsutake (9). 

Oyster Mushrooms (Pleurotus)

As with octenol and hexanal, hexanol is a fatty acid breakdown product.  A top note, hexanol is described as ethereal, fruity, apple, sweet, oily, herbal, green, wine-like; and then it takes a downturn with “slightly goaty” (5). When you smell freshly cut grass, hexanol is one of the things you’re breathing in (25). This one will also be on hand at the conference.  

Hexanol is also found in allspice, apples, bay laurel, bilberry, asparagus, cinnamon, elder flowers, ginger, lavender, orange and violet (5). It’s another popular natural chemical in the flavoring industry and in perfumery (25, 26). It’s being explored as a natural fumigant for fruit due to its antimicrobial effects (27).

It has dose-dependent plant growth stimulating or inhibiting effects depending on the plant (1), again, maybe a way a mushroom influences its surroundings. Hexanol, like octenol and hexanal, acts as a semiochemical transmitting signals among different organisms — fungi, bacteria, plants, insects — in an ecosystem (1). 

Benzoic Acid & Benzaldehyde

Benzaldehyde is a significant contributor to the scent of Oyster Mushrooms (Pleurotus species),  (28), Matsutake (Trichloma matsutake)(29) and the really good-smelling Agarics such as Meadow Mushroom (Agaricus camestris), Horse Mushroom (A. arvensis) and, especially, the Almond Mushroom (A. subrufescens aka A. brasiliensis aka A. blazei…same species based on genetics)(30).  Benzaldehyde is also found in Button Mushrooms and King Boletes (16).

I just ordered a pound of Almond Mushrooms for subjecting hydrosol making and oil infusing. (I’ll likely eat some, too.) Drying does alter the volatile composition of mushrooms, but there should be some good-smelling stuff left. Almond Mushrooms don’t grow in the mountains here in Colorado.  And, well, nothing much did this past season. At least in the Southern Rockies. But you might luck out and find them if you live in Southern Cali. 

Benzaldehyde has a strong scent that is sweet, sharp, bitter, nutty, fruity and maraschino cherry-like, but mostly it smells like almonds. You may have already guessed this based on the name ALMOND Mushroom.  Benzaldehyde is a top note in perfumery (5).  It’s scent and flavor have made benzaldehyde one of the most used chemicals in the flavor industry (31). It’s also got anti-fungal, anti-bacterial and anti-tumor action (31).

Horse Mushroom (Agaricus arvensis)

Benzaldehyde is found in coffee, cinnamon, Rose family members and also in almonds (are you shocked?) (5).  And, here comes the breath thing again…benzaldehyde is also found in our breath (32). (What’s up with this?!)

When benzaldehyde is oxidized, it becomes it’s cousin, benzoic acid. Benzoic acid has a balsamic scent that you’ll be familiar with if you’ve ever worked with benzoin resin or essential oil. Benzoin has long been used as for respiratory conditions and as a topical treatment for wounds.  These uses stem at least in part from the decongesting action and anti-microbial properties of benzoic acid.  Not surprisingly, benzoic acid is in the same mushrooms as benzaldehyde.

Anisaldehyde

Anisaldehyde is another big one in Oyster Mushrooms (33), and is also in Boletes (16) and a bunch of Agaricus mushrooms (4, 34).

Guess what it smells like?  Aside from the obvious, the strong scent of anisaldehyde is also described as balsamic, hawthorn (?!), powdery, sweet, almond, fruity, berry, chocolate, cinnamon, minty, coumarinic, creamy, spicy and vanilla (5). It’s been reported to have a “typical marshmallow flavor” ) (5), but do they mean the real ones or the hooves and corn syrup version? In either event, that’s a lot of descriptors for one isolated chemical. Not surprisingly, it’s popular in the flavor and perfume industries. It’s makes up a significant fraction of some commercial perfumes (5).

Anisaldehyde has both anti-fungal and anti-bacterial activity (35). It may also increase melanogenesis (pigmentation) in cells (36), which helps cells shield themselves from free radical damage caused by radiation.  Aside from the potential use in prevention of skin cancer, the authors of the study posit that anisaldehyde may be helpful for folks with vitiligo or other conditions wherein the skin loses its pigmentation. 

It’s found in aniseed (surprise!), along with star anise, fennel, basil, cinnamon, dill, cumin, tarragon and pine (I wonder if anisaldehyde is why Ponderosas smell like vanilla or butterscotch when you stick your nose up to the bark and sniff).  Black currant, tea leaves (Camillia sinensis) and vanilla also contain anisaldehyde (5).

Methional

Potato.  That’s what this smells like. And tastes like (it’s a major scent and flavor component of baked potatoes).  Methional is an aromatic found in Oyster mushrooms (24), Giant Puffball (37),  Matstutake (9), Button Mushrooms (38), King Bolete (39) and Truffles (40).

Methional is also described as cheesy (especially limburger!), creamy, earthy, brothy, meaty and savory (5). Accordingly, methional activates the umami taste receptor on the tongue (41). Unlike the other aromatics discussed so far, methional contains sulfur. Sulfur-containing molecules tend towards the stinky.  While the odor descriptor would make me inclined to chuck it in the base note file (“meaty” doesn’t exactly make me think “top note”), it’s actually a very small scent molecule and would be expected to evaporate relatively quickly, thus a top note. Next time you want a McDonald’s-scented body spray, reach for methional!

People and other animals tend to like the flavor and scent. Evolution stepped in and switched some plants to animal rather than insect pollinators, with methional being the lure (42). Maybe mushrooms are using it for the same reason…spreading their spores. We humans certainly help by carrying our Bolete bounty along in a basket. 

Methional is also found in asparagus, tamarind, pumpkins, potatoes (duh!) and some fermented foods (5).  It’s an important contributor to the flavor of cheeses, too. 

These are just a small handful of the gazillion aromatics that mushrooms (or their microbial hangers-on) make, with all sorts of surprising scents.  The focus on those here was simply because they’re some of the more common mushroom aromatics.   And they’re found in some of the mushrooms I’m most familiar with in my practice and my kitchen. 

Next time in Plant Healer Magazine (and also in the Mushromatherapy class at this May’s Confluence), we’ll look more into some wonderful smelly mushrooms. Meanwhile — for further geekery —here are links to databases of fungal (and other) aromatics:

References

1. Hung, R, et al (2015) Fungal volatile organic compounds and their role in ecosystems. Appl Microbiol Biotechnol (2015) 99:3395–3405.

2. Song SC, & JM Birmingham (2015) Mushrooms as a source of natural flavor and aroma compounds. Proceedings – 2nd International Conference on Mushroom Biology and Mushroom Products. Chapter 37.  345-66.

3. Patent (2012) Porcini essential oil, essential oil seasoning and preparing method thereof. https://patents.google.com/patent/CN101744209B/en.

4. Moliszewska, E (2014) Mushroom Flavor.  Folia Biologica et Oecologica. 10:80-8.

5. The Good Scents Company Information System http://www.thegoodscentscompany.com/

6. Ito, K & M Ito (2011) Sedative effects of vapor inhalation of the essential oil of Microtoena patchoulii and its related compounds. J Nat Med. 65(2):336-43.

7. Sitkoff, A (2015) Distillations on Ganoderma applanatum. Reishi & Roses.  https://reishiandrosesbotanicals.com/2015/08/10/distillations-on-ganoderma-applanatum/ 

8. Campos, F, et al (2007) Volatile Metabolites From the Wood-inhabiting Fungi Bjerkandera adusta, Ganoderma applanatum, and Stereum hirsutum. J Essential Oil Res. 22(2):116-118.  

9. Cho, IH, et al (2006) Characterization of Aroma-Active Compounds in Raw and Cooked Pine-Mushrooms (Tricholoma matsutake Sing.) J. Agric. Food Chem. 54 (17), pp 6332–6335.

10. Cho, IH et al (2008) Food Chem. Volatiles and key odorants in the pileus and stipe of pine-mushroom (Tricholoma matsutake Sing).  Food Chem. 106(1):71-6. 

11. Bozok, F, et al (2015) Comparison of volatile compounds of fresh Boletus edulis and B pinophilus in Marmara region of Turkey.  Not Bot Horti Agrobo. 43(1):192-5.

12. Nöfer, J et al (2018) The Influence of Drying Method on Volatile Composition and Sensory Profile of Boletus edulis. J Food Quality Article ID 2158482.

13. Kuka, M, et al (2014) Chemical composition of Latvian wild edible mushroom Cantharellus cibarius. Foodbalt. Conference Proceedings. 248-52.

14. Guo, Y, et al (2018)  Characteristic volatiles fingerprints and changes of volatile compounds in fresh and dried Tricholoma matsutake Singer by HS-GC-IMS and HS-SPME-GC-MS. J Chromatogr B Analyt Technol Biomed Life Sci. 1099:46-55. 

15. Gogavekar, SS, et al (2014) Important nutritional constituents, flavour components, antioxidant and antibacterial properties of Pleurotus sajor-caju. 51(8):1483-91. J Food Sci Technol. 51(8): 1483-91.

16. Rapior, S, et al (1997) Volatile aroma constituents of Agarics and Boletes. Recent Res Dev Phytochem. 1:567-84.  

17. Zhuang, H, et al (1996) The Impact of Alteration of Polyunsaturated Fatty Acid Levels on C,-Aldehyde Formation of Arabidopsis thaliana Leaves. Plant Physiol. 11 1 : 805-812.

18. Gardner, HW, et al (1990) Hexanal, trans-2-hexenal, and trans-2-nonenal inhibit soybean, Glycine max, seed germination. J. Agric. Food Chem. 38 (6):1316–1320.

19. Lanciotte, R, et al (2003) Application of hexanal, (E)-2-hexanal, and hexyl acetate to improve the safety of fresh-sliced apples. J Agric Food Chem. 51(10):2958-63.

20. Song, J, et al (1996) Hexanal vapor is a natural, metabolizable fungicide: Inhibition of fungal activity and enhancement of aroma biosynthesis in apple slices.  J Am Soc Hort. 121(5): 937-42.

21. Patent application (2015) Antiviral activity from medicinal mushrooms and their active constituents. Justia Patents. https://patents.justia.com/patent/9931316 

22. Francke, W, & S Schultze (2010) Pheromones of Terrestrial Invertebrates. Comprehensive Natural Products II. Chemistry & Biology. 4:153-223. Elsevier.  

23. Ionescu, R, et al (2011) Detection of Multiple Sclerosis in exhaled breath using bilayers of polycyclic aromatic hydrocarbons and single wall carbon nanotubes. ACS Chem Neurosci. 2(12): 687–693. 

24. Usami, A, et al (2014) Chemical composition and aroma evaluation of volatile oils from edible mushrooms (Pleurotus salmoneostramineus and Pleurotus sajor-caju). J Oleo Sci. 63(12):1323-32.

25. 1-Hexanol. PubChem Open Chemistry Database https://pubchem.ncbi.nlm.nih.gov/compound/1-Hexanol#section=Top    

26. 1-Hexanol, FooDB http://foodb.ca/compounds/FDB008072

27. Hamilton-Kemp, TR, et al (1996) Metabolism of natural volatile compounds by strawberry fruit. J. Agric. Food Chem. 44 (9), pp 2802–2805.

28. Beltran-Garcia, M, et al (1997) Volatile Compounds Secreted by the Oyster Mushroom (Pleurotus ostreatus) and Their Antibacterial Activities

J. Agric. Food Chem. 45 (10), pp 4049–4052. 

29. Li, Q, et al (2016) Chemical compositions and volatile compounds of Tricholoma matsutake from different geographical areas at different stages of maturity. Food Sci & Biotechnol. 25(1):71-7.

30. Agaricus blazei, Agaricus brasiliensis, Himematsutake http://www.medicalmushrooms.net/agaricus-blazei/.

31. Verma, RS et al (2017) Natural benzaldehyde from Prunus persica (L.) Batsch. Int J Food Prop. 20(52):S1259-63.

32. Clegg, B (2018) Benzaldehyde. Chemistry WorldRoyal Society of Chemistry. https://www.chemistryworld.com/podcasts/benzaldehyde/3009422.article.    

33. Okamoto, K, et al (2002) Biosynthesis of p-anisaldehyde by the white-rot basidiomycete Pleurotus ostreatus. J Biosci Bioeng. 93(2):207-10.

34. Rapior, S, et al (2002) The anise-like odor of Clitocybe odora, Lentinellus cochleatus and                                           Agaricus essettei. Mycologia. 94(3):373-6.

35. Shen, H-S, et al (2017) Antimicrobials from mushrooms for assuring food safely. Comp Rev Food Sci Food Safety. 16:316-329.

36. Nitoda, T, et al (2007) Anisaldehyde, a Melanogenesis Potentiator. Z Naturforsch. 62(1-2):143-9.

37. Leffingwell, JC & ED Alford (2011) Leffingwell Reports, Vol. 4. Feffingwell & Associates. Alford Consulting.

38. Qin, L, et al (2011) Effect of different cooking methods on the flavour constituents of mushroom (Agaricus bisporus (LangeSing) soup. Food Sci & Technol. 46(5):1100-08.

39. Guedes de Pinho, P, et al (2008) Aroma compounds in eleven edible mushroom species: Relationships between volatile profile and sensorial characteristics. CIMO – Artigos em Proceedings Não Indexados à WoS/Scopus

40. Vita,F, et al (2015) Volatile organic compounds in truffle (Tuber magnatum Pico): comparison of samples from different regions of Italy and from different seasons. Sci Reports. 5: Article number: 12629.

41. Toda, Y, et al (2018) Positive/Negative Allosteric Modulation Switching in an Umami Taste Receptor (T1R1/T1R3) by a Natural Flavor Compound, Methional. Sci Rep. 2018 Aug 7;8(1):11796.

42. Wester, P, et al (2019) Scent chemistry is key in the evolutionary transition between insect and mammal pollination in African pineapple lilies. New Phytol. doi: 10.1111/nph.15671.

Monkshood

Went looking for mushrooms today. Found nothing but a few Corts : ( But I did find a lovely patch of Monkshood (Aconitum columbianum), one of my favorite wildflowers (for just looking at!) around here.

Monkshood contains alkaloids such as aconitine, mesaconitine, and related molecules the toxicity of which manifests in the nervous system and heart, with some GI stuff as well…vomiting, nausea, diarrhea. Aconitum species do have a history of use in Traditional Chinese Medicine and elsewhere after very careful processing and with very careful dosing. (Even handling the plant can result in some neurotoxic effects.) It is deadly in high enough dosage and is not a plant to experiment with.


In other words, don’t stick it in your mouth. Just look at it and marvel at its beauty

A Primer on SARS-CoV-2 & things to do while stuck at home

 

SARS-CoV-2. Micrograph from NIAID

First, where I’m coming from… There is the “Germ Theory” in which pathogenic microbes cause infectious disease.  Then there’s the “Terrain Theory” in which the “terrain” a person provides is the determining factor in disease, with the microbes being a symptom of diseased tissues as well as a provacateur of further disease. As a former virologist/infectious disease researcher and a current herbalist, I feel that both pathogens and terrain are important, with one or the other being more dominant, depending on the situation.

On one hand, folks lucky enough to be in a healthy environment and who practice healthy eating and living habits are generally less susceptible to infectious diseases;  while someone who is unhealthy for various reasons may wind up with issues due to a microbe that would otherwise be harmless (eg. Candida). On the other hand, there are multiple pathogenic microbes that will infect and cause disease in healthy people. Hantavirus, certain influenza strains, and ebola virus, are some (extreme) examples of this.  So,  I take the middle road…

~~~

WHAT ARE CORONAVIRUSES?

  • A family of viruses that infect birds and mammals (including us, obviously!)
  • Coronaviruses are called such because the bits that stick out from the spherical viral particle resemble the Sun’s corona, or outer layer.
  • Several coronaviruses cause upper respiratory tract infections in people and are one of the causes of the common cold
  • “Betacoronaviruses” are part of the coronavirus family and include the currently  circulating SARS-CoV-2 virus, the SARS-CoV virus that caused the SARS epidemic in 2003 and MERS-CoV that caused a small but severe outbreaks from 2012-2014.

SARS-CoV-2 & COVID-19

  • SARS-CoV-2 is the name of the virus causing the current    pandemic
    • “SARS” is for Severe Acute Respiratory Syndrome        SARS-CoV-2.  Image from NIAID
    • “CoV” is for coronavirus                                                                                                                                                                           
    • “2” is because this is highly related to SARS-CoV that caused the SARS epidemic (their genetic code is ~ 80% identical).
  • COVID-19 is the name of the disease that SARS-CoV-2 causes:
    • “COVI”for coronoavirus
    • “D” for disease
    • “19” for 2019, when it emerged (the first case was identified on Dec 1st

Spread of SARS-CoV-2

  • The increased case numbers being reported here in the US are due both to viral    spread but also to increased testing identifying previously unknown cases.
  • The 1st case was reported on Dec 1st, 2019. As of March 29th, there were 715,076 identified cases of COVID-19 worldwide. The number is likely higher simply because not everyone is being tested. Mildly symptomatic or asymptomatic infections are likely being missed. 
  • Estimates are that an infected person on average infects 2-2.5 other people. Based on this, you can see how this can greatly increase the number of cases over time. If those 2 people then spread it to 2 more people each, etc… This is one of the reasons that social distancing has been evoked. If folks aren’t out mixing with a bunch of other people, it reduces the odds of transmission.
  • SARS-CoV-2 is spreading much more than SARS-CoV did in the 2003 SARS epidemic. Recent research suggests that this may be due to a surface protein that differs slightly between the 2 viruses. This difference may allow SARS-CoV-2 to replicate not only in the lungs as did SARS-CoV, but possibly also in the upper respiratory tract and maybe even in GI tract as well https://www.medrxiv.org/content/10.1101/2020.03.05.20030502v1 This report is a preprint, meaning that the research hasn’t yet been peer reviewed.
  • There is evidence from the above small study that viral shedding can be as earlier as the 1st week of symptoms, and others note it may also be happening in the presymptomatic phase https://science.sciencemag.org/content/early/2020/03/24/science.abb3221  So, even if you feel fine, you should still be practicing hygienic techniques like social distancing, frequent hand-washing, disinfecting surfaces, covering your mouth/nose (not with your hand!) when coughing or sneezing.
  • The same study cited above found that aerosolized virus (the form in viruses spread from coughing or sneezing) can last in the air an average of ~ an hour, with the upper end of range being ~2.5 hours. This and the surface survival data exemplify why it’s important to cover one’s mouth when coughing or sneezing, preferably with a tissue or the crook of your elbow (and not your hand). Don’t forget to regularly disinfect handheld devices….phones, tablets, etc.

More on COVID-19

  • A recent research review puts the worldwide average case fatality rate at 2.2% https://onlinelibrary.wiley.com/doi/full/10.1111/tmi.13383 Tho the WHO has it at 3% as of March 3.   This means that approximately 2-3 out of every 100 people infected die.  But estimates  this vary from country to country. In Italy, it has been estimated as high as 10%, while in the US it’s less than 1.5%, and in Germany it’s 0.5%. What these numbers actually represent is complicated, as they’re influenced by who is being tested, by availability of health care, and other factors. As more are tested, these rates may turn out to be lower than current reports.
  • Symptoms emerge on average ~5 days post-infection but may show up sooner or even out to 2 weeks post-infection. The most common symptoms:  Fever (often low), cough (often dry at first) and fatigue. Others include headache, diarrhea, low white blood cell count; and at the more serious end of the spectrum, difficulty breathing, lung damage/pneumonia-like effects, haemoptysis (coughing up blood), and cardiac injury. Loss of taste and smell is coming out in anecdotal reports.
  • Elderly folks or those with chronic health issues (eg. lung, heart) are those most at risk for severe disease

SOME PERSPECTIVES

  • As of 27 March, COVID-19 has killed over 33,300 people globally, with the rate here in the US surpassing 1,200.  As mentioned, the average global case fatality rate is somewhere on average around 2-3% at this point (higher in the elderly). 
  • For comparison, the case fatality rate of the average seasonal flu is about 0.1%. That for SARS was about 10% and MERS was a whopping 34% (though both infected far fewer people than the current pandemic virus).
  • Though it has a lower case fatality rate, this season’s flu has infected over 34,000,000 people in the US, with over 16,000 deaths
  • 2000 children die daily, worldwide, from diarrhea
  • This all is not to belittle the current serious pandemic but to put it into context. We don’t panic about seasonal flu and we shouldn’t panic about this. Instead, we should treat it with respect and take the appropriate precautions being put out by organizations such as the WHO.
  • Because this is a new virus in humans, there are a lot of significant unknowns, and this along with the case fatality rate can generate a lot of fear. Coupled with stretched hospital resources, and emergency departments being in triage mode, this means that this is a serious situation. But freaking out and hoarding aren’t helpful.

TESTING

  • There are a variety of approaches being used to test for infection with SARS-CoV-2 with pros and cons for each.

MEDICAL TREATMENT

  • At this point, it’s supportive.
  • There are existing drugs that are currently under investigation for repurposing, but none are recommended for use as yet, pending results on efficacy and safety.  This is despite what you may hear on the news.
  • There is no vaccine. Efforts are underway. But development of a vaccine for the similar SARS-CoV ran into some hurdles (there still isn’t one commercially available to my knowledge). For one, there is a quirk of the immune response to some viruses that can make vaccine development challenging. Also, given the relatively limited extent of the SARS epidemic, there ultimately wasn’t a lot of interest in funding clinical trials. So, in terms of a vaccine for SARS-CoV-2, it’s not going to be here tomorrow or next month or…  Though many labs are working on it. More details: http://www.virology.ws/2020/03/11/sars-cov-2-the-vaccine-landscape/

HERE IS A SITE PROVIDING MORE DETAILS ON THE PANDEMIC (that is updated regularly)…        https://arstechnica.com/science/2020/03/dont-panic-the-comprehensive-ars-technica-guide-to-the-coronavirus/

SO, WHAT CAN YOU DO WHEN UNDER A “STAY THE HECK AT HOME” ORDER?

  • Use stress-relieving techniques. Whatever works for you:  Sitting and focusing on your breath. Music. A good book. Start turning your soil for a garden if lucky enough to have a yard. Go for a walk in nature (outdoor exercise is still allowed in CO as long as you are maintaining a safe distance from others).  This is for quality of life and also because chronic stress may disrupt healthy immune system function. Keep in mind that if you have any symptoms as all, it is critical that you stay in to prevent spread.
  • Get some fresh air (not in a crowd) – Research during the 1918 flu pandemic found that folks who had access to fresh air fared better than those who were cooped up.  Get out on your porch, balcony, etc, at least for a few minutes daily.
  • Connect – Especially if you live alone. Call loved ones. Even use the dreaded internet to connect.  Even if you aren’t hugging anyone at the moment, simply hearing a voice can be enormously supportive.  If you need supplies but can’t get out, ask someone.
  • Disinfect – When you need to go out for anything, going to the store or other necessary trips, use disinfectant on your hands while out, wash your hands thoroughly with soap (regular ol’ soap is fine) and don’t touch your face. Mind you, hand washing and not touching your face is most important of all. 
      • For example, as soon as I get back to my car, I use a spray of 70% ethanol and some essential oils. (This level of alcohol is sufficient, but I like adding some essential oils for scent, about 8 drops per ounce). You can use this on your steering wheel, door knobs or any other surface not damaged by alcohol.   
      • You can also use chlorine bleach (sodium hypochlorite) diluted with water as a surface-cleaning solution as long it’s not on a surface it will damage – 4 teaspoons bleach per quart of water as per the CDC. Make sure the bleach hasn’t passed its expiration date.
  • Herbs – here’s a blurb I posted on FB recently on stuff you may have around the house or yard. Keep in mind, this is simple support for your respiratory and immune systems to help you stay healthy, and is not a substitute for social distancing, hand-washing and using disinfectants to keep your space clean.

~ ~ ~

Given that a lot of folks are stuck at home, now’s a good time for tea making. Here are a few ways to make an aromatic tea to help keep you healthy using stuff you may have in your kitchen, yard or other nearby outdoor space….  A few ideas:

– Pine/Fir Needles and Citrus Peel – finely chopped      

– Thyme and Citrus Peel

 

– Marjoram and Citrus Peel

– Rosemary, Sage and Citrus Peel

– Ginger, pinch of Cinnamon, a couple Cloves – skip this one if you’re feeling dry

Add Mint (Spearmint, Peppermint, Lemon Balm, Catnip, etc), a pinch of Black Pepper and/or several finely chopped, ripe Juniper berries to any of the above if you’ve got them available. For flavor, add a small amount of local, raw honey after steeping.  A couple/few cups a day. Switch your blend up to keep it interesting.

Note: A medicinal tea is strong. If any of the botanicals here result in a wimpy, mildly-flavored brew, then steep it longer or add more herbs. Or, it could be that your Thyme or what-have-you herbs have been in the spice cabinet for too long.  Dry herbs (other than roots, seeds and dried berries) maintain their properties for only about a year if stored properly…

Here is a link to tea-making instructions. Use the “hot infusion” method described: https://basmati.com/…/witchin-kitchen-3-methods-making-medi…

~ ~ ~

  Steams with aromatic plants are a great way to provide a pleasant atmosphere  when stuck at home.  Use what you have on hand. Mint, Rosemary, Marjoram, Thyme, Cloves, Cinnamon, Allspice, Ginger, Citrus peels, etc. I love Pine or Fir Needles as well as Juniper Leaves/Berries. Simmer a couple of handfuls in water on the lowest setting. (Don’t leave them unattended or you may wind up with a bunch of firefighters at your door. Ask me how I know).  Yes, you can do the same with essential oils or using them in a diffuser, just don’t diffuse all day…you can overdo it.  But I find the scent to be richer and more complex using the plant bits themselves. 

© 2020, Anna Marija Helt, PhD, Osadha Natural Health, LLC  

Stuck at home? Here are some teas to make…

Given that a lot of folks are stuck at home, now’s a good time for tea making. Here are a few ways to make an aromatic tea to help keep you healthy using stuff you may have in your kitchen, yard or other nearby outdoor space….
 
 
(use the ‘hot infusion” method from the article for the following)
 
A few ideas:
 
Pine or Fir Needles and Citrus Peel – finely chopped
 
Thyme and Citrus Peel – can add in several finely chopped Juniper berries
 
Marjoram and Citrus Peel
 
Rosemary and Citrus Peel
 
Can add Mint (Spearmint, Peppermint, Lemon Balm, Catnip, etc) to any of the above. And any of the above ingredients can be used in combination or alone. For flavor, add a small amount of local, raw honey after steeping.
 
Note: A medicinal tea is strong. If any of the botanicals here result in a wimpy, mildly-flavored brew, then steep it longer or add more herbs. Or, it could be that the Thyme/etc have been hanging out in your spice cabinet for too long. Dry herbs other than roots and seeds maintain their properties only for about a year if stored properly…