How does the surrounding environment influence the effectiveness of medicinally active compounds found in herbal medicine?

1. Chemical Ecology Can Help
Find Good Medicine
Dr. Michael Tims

2. Modified from Bruinsima & Dicke 2008
Shoots
Flowers
Roots
Pathogens
Pathogens
Herbivores
Pollinators
Carnivores
Competitor
plants
Secondary
carnivores
Symbionts
Parasitic plants
Carnivores
Herbivores
Plant Defense:
Multitrophic
Interactions

3. Kin Recognition
• Roots grow more in proximity to genetically related plants
(Bhatt)
• Recognition of kin based on root secreted chemicals
(Biedrzycki).
• Plants also react to kin in the aerial portions
• reorient leaf growth when growing near kin, but not
near unrelated plants of same species
• produce more seeds interacting with kin vs. nonkin
(Crepy and Casal)

4. Allelopathic Function
• Influence or effect of one living
plant upon another
• Juglone released into
environment inhibits growth of
other plants
• Root to root contact must exist
for toxicity to occur
juglone
Black walnut (Juglan nigra)

5. Phytoalexin (Defense Function)
• Infection of grape vines by the fungus
Botrytis cinerea results in the
production of phenol, resveratrol
• Responds to damage cues
• 60 to 140 µg/ml inhibits fungal spore
germination
~0.0002 teaspoons/Liter
(Hoos and Blaich, 1990)
resveratrol
Vitis vinifera

6. Endophytes
• Camptotheca acuminata (happy tree)
• Camptothecin - quinoline alkaloid
• Anticancer compound - inhibits topisomerase I (DNA
replication enzyme)
• Fusarium solani (fungal endophyte) produces precursor
• Plant enzyme able to make final product
• Accumulate in crystals in the vacuole of specialized cells
in epidermal tissues, as well as glandular trichomes
• When grown alone, plant can’t make camptothecin
(Kursari et al. 2012)

7. Below Ground

8. Mycorrhizal Fungi
Symbiosis: close biological association of different
species, often for mutual benefit

9. Mycorrhization
• Plant root supplies sugars to the fungus
• Induction of JA biosynthetic enzymes
• Levels of jasmonate ↑ leading to …
• … enhanced accumulation of soluble sugars in plant root
• … enhanced plant defense

10. Artemisia annua
Inoculation with mycorrhizal fungus, Glomus mosseae,
increased yield artemisinin, a sesquiterpene lactone .
(Awasth, A. et al. 2011)

11. Influence on Phenolic Compounds
• Gabriele et al. (2016) investigated the effect of
mycorrhizal inoculation of various Sangiovese wine
grapes
• Found fungi increase 14 polyphenols compared to
uninoculated plants

12. Phenolic compounds
Benzene ring
Cyclohexane structure – three double
bonds. The basis of all ‘aromatic’ chemicals
Hydroxyl group (OH)
One or more attached to benzene ring
Functional groups (= aryl groups)
Methyl CH3
Methyoxy CH3O
Amino NH2
Carboxyl COOH
Lactone ring Coumarins
CO2H
OH
Salicylic acid

14. Metabolism and Geography
Metabolism Compounds Geography
Shikimic acid Lignans
benzylisoquinoline alkaloids
Forests –
greater
biomass
Acetic acid
Acetate
Steroids
pyrrolizidine alkaloids
Dry Grasslands
Ecological
boundary or
Ecotone
Creative potential -
interruptions of chemical
gradients
(Albuquerque et al. 2012)
Based on data from Brazil

15. Flavonoids
• Accumulate at root tips/cap
• Large portion of root exudate
• Easily modified
• Biosynthesis triggered by transcription factors  role as
elicited signal compounds
• Trigger mycorrhizal fungi to explore rhizosphere
(Hassan and Mathesius, 2012)

16. Ecological Infuences on
Ginsenosides
• Total ginsenosides in roots decline after mid-September
(Li and Wardle, 2002)
• Highest content:
root hairs > lateral roots > cortex > interior taproot

17. Protopanaxadiols Protopanaxathiols
Tachikawa et al. (2003)

18. Ginsenoside by Species and Source
Higher levels
diols triols
P. quinquefolia Rg1
wild Rg1 Re
cultivated Rb1
P. ginseng Rb1 Rd
wild Rg1 Re
cultivated Rb1 Rb2
• Greatest chemotype variation in wild populations
• Ratio differences of woods grown vs. cultivated ginseng
explained greater brain neuronal activity inhibition
(Yuan et al. 2001)
Zhu et al., 2004
Wang et al., 2010

19. Wild Sourced Leaf vs. Root
(NC Collection)
• No relationship between age and ginsenoside content
• Rg1:Re ratio (triols) used by industry
• Re absent from numerous samples
(James M. 2013)
Higher in Leaf Higher in Root
Total ginsenosides Rb1(diol) and Rg1 (triol)
Rb2 and Rd (diols)

21. Above Ground

22. Plant Response to Herbivores
Plant Defense Traits

23. Leaf Volatile Signals
(Consuelo M. et al. 2000)
Methyl Jasmonate

24. Same Moth
A1) daytime volatiles
A2) nighttime volatiles
Different Moths
B1) nighttime volatiles
B2) nighttime volatiles
C) mechanical damage

25. Why does Night Herbivory Inhibit
Female Moths from Ovipositing?
Damage - cue emitting signal
• More competition for offspring
• Greater population of predators attracted by chemical
cues
• Direct defense induction increases the likelihood of
plant toxins being present

26. Monoterpene
Synthesis in Thymus
vulgaris
Chemotypes
Thymus vulgaris ct. linalool
Thymus vulgaris ct. thymol
Thymus vulgaris ct. carvacrol
Thymus vulgaris ct. gerinol
Thyme vulgaris ct. thuyanol

27. Light Exposure and Elderberry
• Sambucus nigra subsp. canadensis
• Wild elderberry fruits sampled over two years in eastern
US
• Profiled select flavanols (quercitin, isoquercitin, rutin) and
chlorogenic acid
• All metabolites higher in south, particularly interior
(Mudge, E. et al. 2016)

28. Seasonal Influence on Green Tea
• Spring (dry) vs. Monsoon season
• Metabolite concentrations that increase greatly in Spring
• Monoterpenes
• Diterpenes
• Acids
• Aldehydes
• Esters
• Ketones
• Methyl salicylates
• Methyl jasmonates
(Kowalsick et al. 2014)

29. • Growers using sensory analysis rate spring harvest a
higher quality than monsoon rain season harvest
• Stronger aroma
• Better Taste
• Corresponded with higher concentration of metabolites in
Spring vs. Monsoon season
• Catechins (especially EGC, EGCG, ECG)
• Methylxanthines (caffeine, theobromine, theophylline)
(Ahmed, S et al. 2014)

30. • EGCG concentrations increased with age and leaf quality
• Caffeine concentrations greater in younger leaves

31. Processing Influence on Flavan-3-ols
%
Flavan-3-ol
Total phenolics
 71% to 3%
(Del Rio et al. 2004)
Fresh to processed green tea increases
EGCG levels
(Lin et al. 2003)

32. Ecological Role of Flavan-3-ols
Induced response:
• Wounding (Feucht et al. 2004)
• Plant-plant interactions (Bais et al. 2003)
• Fungal elicitation increases level of epi-gallated flavan-
3-ols and degree of polymerization of flavan-3-ols
• Cross linking of excreted fungal enzymes (Skadhauge
et al. 1997)

33. Challenge of Primary Cell Walls
Extraction matrix created conditions where EGCG bound to
proteins and lipids during storage
http://www.ccrc.uga.edu/~mao/intro/ouline.htm

34. Optimized Extraction

35. Structural Features of Flavan-3-ols
Fungal elicitation increases level of epi-gallated flavan-3-
ols and degree of polymerization of flavan-3-ols
(+)-catechin (C)
HO O
OH
OH
OH
OH
(-)-epicatechin (EC)
OHO
OH
OH
OH
OH
(-)-epigallocatechin (EGC)
OH
HO O
OH
OH
OH
OH
O
O
C
O
OH
(-)-epigallocatechin gallate (EGCG
OH
OH
OH
OH
OH
OH
HO
galloyl ester
(trans-) (cis-)
epimers

36. Stability During Storage
T = 0 h

37. Stability During Storage
T = 24 h

38. Echinacea purpurea Cultivar
Screening
Trout Lake Farm
• Mechanical harvesting
• Flower heads, size and uniformity of flowering time
• Large smooth leaves (leaf hairs capture dirt)
• Deep purple color
• Intense tongue tingle
(Letchamo et al. 1999)

39. • Field of 2 million identified 420 optimal plants
• Used organoleptic and HPLC analysis to select 360
propagation lines
• 20-30 of each line replanted in three different locations
• Used phytochemical analysis on the aerial parts to
analyze at 4 stages of flower development

40. Cichoric acid
Isobutylamides

41. Parasitic Plants

42. (Estabrook, EM. and Yoder, JI., 1998)

43. Pedicularis
• > 40 species in North American alone
• World wide range of Pedicularis host plants ~80 different
plant species (35 families)
• Clinical efficacy inconsistent
(Piehl 1963)

44. Host Chemical Influence
Parasitic Plant Host Alkaloid type
P. bracteosa
P. groenlandica
Senecio triangularis Pyrrolizidine
(senecionine)
P. bracteosa Picea engelmannii Piperidine
(cis-pinidinol)
P. crenulata. Thermopsis montana Quinolizidine
(anagyrine)
P. grayi Thermopsis divaricarpa Quinolizidine
(N-methylcytisine)
P. semibarbata L. fulcratus Quinolizidine
(α-isolupanine)
(Schneider and Stermitz 1990)

45. Ghost Pipe
• Monotropa uniflora (Ericaceae) - blueberries, cranberries,
heath, Rhododendron, azaleas, Arctostaphylos, and
Arbutus
• Many other species of Monotropa
• Other mycoparasitic plant genera of the subfamily
Monotropoideae - Pterospora, Hermitomes, Sarcodes,
Pityopus

46. • 13 Montropa phylogenetic lineages identified that have a
specialized relationship to different mycorrhizal genera
• No overlap in mature plant/fungal symbionts even if fungi
and the plants share closely related geographical patterns
(Bidartondo and Bruns 2001)
• Clinical efficacy consistent?
• Fragility of plant populations

47. Questions?
mtims@muih.edu