1 of 33

A systematic evaluation of Eremophila phytochemistry

Dr Ben Greatrex, 2020

The University of New England

2 of 33

Natural products as drugs (1985-2001)

3 of 33

The genus Eremophila

3-5 million years old – rapid speciation
284 named species in the Australian plant name index (APNI)
Only ~80 have been examined for phytochemicals
Extremophiles living in arid lands and deserts
Wide variety of natural product classes

Distribution of Eremophila coded by species

E. maculata

E. bowmannii

E. spectabilis

CC BY 4.0, Geoff Derrin, Wikimedia commons

CC BY 2.5, Melburnian, Wikimedia commons

E. brevifolia

4 of 33

Eremophila species lacking with phytochemical studies
E. accrescens	E. decussata	E. magnifica	E. revoluta
E. adenotricha	E. delisseri	E. maitlandii	E. rhegos
E. annosocaulis	E. demissa	E. malacoides	E. rigens
E. anomala	E. dendritica	E. margarethae	E. rigida
E. appressa	E. densifolia	E. micrantha	E. saligna
E. arachnoides	E. deserti	E. muelleriana	E. santalina
E. arbuscula	E. dichroantha	E. obliquisepala	E. sargentii
E. arenaria	E. divaricata	E. oblonga	E. scaberula
E. arguta	E. elderi	E. obovata	E. scrobiculata (new species)
E. attenuata	E. enata	E. occidens	E. serpens
E. aureivisca	E. eriocalyx	E. ovata	E. setacea
E. barbata	E. eversa	E. paisleyi	E. shonae
E. ballythunnensis (new species)	E. fallax	E. pallida	E. simulans
E. battii	E. fasciata	E. pantonii	E. spathulata
E. behriana	E. ferricola (new species)	E. papillata	E. spinescens
E. bowmanii	E. flabellata	E. parvifolia	E. splendens
E. brevifolia	E. gibbifolia	E. pendulina	E. spongiocarpa
E. buirchellii (new species)	E. glandulifera	E. pentaptera	E. spuria
E. caerulea	E. goodwinii	E. perglandulosa	E. stenophylla
E. caespitosa	E. graciliflora	E. petrophila	E. strongylophylla
E. calcicola (new species)	E. gracillima	E. phillipsii	E. subangustifolia (new species)
E. campanulata	E. grandiflora	E. physocalyx	E. subfloccosa

5 of 33

E. canaliculata	E. hamulata (new species)	E. pilosa	E. subteretifolia
E. caperata	E. hillii	E. pinnatifida	E. succinea
E. capricornica (new species)	E. hispida	E. platythamnos	E. tenella
E. chamaephila	E. humilis	E. polyclada	E. ternifolia
E. christophori	E. hygrophana	E. praecox	E. tetraptera
E. ciliata	E. incisa	E. prostrata	E. tietkensii
E. citrina	E. jamesiorum (new species)	E. psilocalyx	E. undulata
E. clavata	E. jucunda	E. pterocarpa	E. veneta
E. coacta	E. koobabbiensis	E. punicea	E. vernicosa
E. compacta	E. laanii	E. purpurascens	E. veronica
E. compressa	E. labrosa	E. pusilliflora (new species)	E. verrucosa
E. conferta	E. laccata (new species)	E. pustulata	E. victoriae (new species)
E. congesta	E. lachnocalyx	E. ramiflora	E. viscimarginata
E. conglomerata	E. lactea	E. recurva	E. warnesii
E. cordatisepala	E. lanata	E. regia (new species)	E. weldii
E. crassifolia	E. lanceolata	E. resiliens (new species)	E. willsii
E. cryptothrix	E. lehmanniana	E. resinosa	E. yinnetharrensis(new species)
E. cuneata	E. macgillivrayi	E. reticulata	E. youngii
E. daddii (new species)	E. mackinlayi	E. retropila

6 of 33

Natural products in the genus Eremophila

Iridoids

Tubercles

Muurolanes

Cedranes

Eremophilones

Serrulatanes

Viscidanes

10 carbon

15 carbon

20 carbon

Terpene

Sesquiterpene

Diterpene

7 of 33

Techniques for characterisation

Gas chromatograph

Nuclear magnetic resonance

spectrometer

8 of 33

A database of volatiles

Terpenes

Mainly sesquiterpenes

Gas chromatograms

9 of 33

Compound identification

10 of 33

235 species in database

11 of 33

A monograph for E. alternifolia

12 of 33

Aged vs Fresh material (E. bignoniiflora)

13 of 33

1. Eremophila longifolia

One of the most important traditional medicines
Validated history of traditional use for ceremonial and medicinal purposes
Highly variable with regards to chemotypes
Some geographical separation of chemotypes

Distribution of E. longifolia

CC BY 2.5, Melburnian, Wikicommons

14 of 33

Smoking ceremony

Smoking ceremonies follow childbirth and also circumcision
Green leaves are placed upon hot coals to produce a steam distillate

15 of 33

Pyrolysis versus extraction

Solvent extract

Pyrolysis

	S. aureus	S.epiderm	B. subtilis	S. typh	C. albicans
Acetone	8.75	8.75	4.4	>	8.75
H₂O	>	>	>	>	>
pyrolyis	0.3±0.01	2.3±0.15	0.3±0.12	1.1±0.05	2.3±0.2
Furan	0.5±0.1	0.25±0.15	0.25±0.5	0.25±0.5	0.13±0.05
Tetra (μg/ml)	0.25-2.8	0.4-0.5	0.25-0.13	0.4-0.5	-
Nyst (μg/ml)	-	-	-	-	1.5-3.4

GCMS trace

Antimicrobial activity (mg/mL)

16 of 33

Furan isolation and characterisation

17 of 33

Presence of furan in chemotypes

Voucher	Chemotype	EO yield %	Furan
1	Limonene, α-pinene, α-terpinolene	0.02-0.1	++++
2	Limonene, α-pinene	0.02-0.1	+
3	Limonene> α-pinene	0.02-0.1	+++
4	Limonene>α-pinene, borneol	0.02-0.1	++++
5	α-Pinene>limonene	0.02-0.1	++++
6	Karahanaenone	0.5	-
7	Karahanaenone	0.02-0.1	++++
8	Isomenthone/menthone	3.5-8	Trace
9	Safrole/methyl eugenol	5.5	-
10	Fenchone	0.5	++++
11	Fenchyl acetate	1	++++
12	Fenchyl acetate	0.1-0.2	Trace

Occurrence of furan in either the essential oil or hydrosol of chemotypes

of E. longifolia, produced by hydrodistiallation of leaves with detection by GCMS.

18 of 33

Furan biogenesis

Smoking method used traditionally is important for activity

19 of 33

2. E. Mitchellii E. Sturtii

20-40 μmol against P. falciparum

10 mg 28 mg 2 mg

20 of 33

E. Sturtii

21 of 33

Mitchellene F Mitchellene H

J. Nat. Prod. 2018, 81, 405−409. J. Org. Chem. 2019, 9637-9647

E. Sturtii

22 of 33

E. Sturtii E. Mitchellii ?

�

Voucher specimen (73007710) from Liu et al. (Liu, Q.; Harrington, D.; Kohen, J. L.; Vemulpad, S.; Jamie, J. F. Phytochemistry 2006, 67 (12), 1256–1261).

Provided by the Downing Herbarium (Macquarie University). Redetermined as Eremophila mitchellii.

J. Nat. Prod. 2018, 81, 405−409.

23 of 33

E. mitchellii E. sturtii

E. sturtii E. mitchellii

mitchellii

24 of 33

25 of 33

2. Eremophila dalyana

Aromatic shrub used for the treatment of colds and Sarcoptes scabiei infestation

Used traditionally by the Eastern Arrernte and Alyawarr peoples of Central Australia

26 of 33

3. Eremophila Dalyana chemotypes

	NT	QLD
Yield % w/w	0.93	0.95
Eucalyptol	9.7	1.6
γ-Terpinene	0.9	-
Myodesert-1-ene (1)	21	96
Terpinen-4-ol	1.4	-
cis,cis-Nepetalactone	3.1	-
Elemol	26.2	1
8-epi-γ-Eudesmol	0.9	-
γ-Eudesmol	6.5	-
β-Eudesmol	4.7	-
α-Eudesmol	6	0.8

27 of 33

	NT1	NT2	NT3	QLD1	2	3	EE	+Control^*
S. aureus	500±500	500±500	130±75	1000	500±500	1000	20±15	0.5
S. epidermidis	1000	-	-	-	-	-	-	0.5
S. typhimurium	1000	-	-	-	-	1000	-	0.5
P. aeruginosa	-	-	-	-	-	-	-	32
E. coli	1000	1000	1000	1000	-	-	-	1
B. subtilis	125±80	60±30	60±30	-	-	-	1±0.5	1
C. albicans	500±500	250±100	60±30	750±350	1000	1000	130±40	4

Antimicrobial activities (μg/mL) of essentiol oils and isolated components.

3. E. Dalyana antimicrobial activity

Control = tetracycline, EE = elemol and eudesmol isomers 5:1:1:1

28 of 33

3. E. Dalyana confirmation of structure

29 of 33

4. E. complanata, E. nivea x E. drumondii

Species	S. aureus	S. epidermidis	B. subtilis	P. aeruginosa	E. coli
E. complanata	250-333	63-125	125-208	>500	>500
E. nivea × E. drummondii	250-333	83-125	125-333	>500	>500
Tetracycline	0.13-0.25	32-64	0.063	8-16	1-8

MICs of active fractions (µg/ml)

A screen of >100 species of Eremophila for antimicrobial activity showed activity in 4 species. E. complanata, E. drumondii, E. nivea and a hybrid.

E. complanata

E. nivea

Fitoterapia. 2018, 126, 29-34.

30 of 33

4. E. complanata, E. nivea x E. drumondii

	1a		2a
Pos.		¹³C	¹³C	Dδ ¹³C_1a-2a
1		27.8	27.8	0.0
2		27.2	27.2	+0.09
3		19.7	19.8	-0.06
4		42.0	42.0	0.0
5		120.9	120.9	-0.06
6		108.6	108.7	0.03
7		145.8	145.9	0.0
8		141.9	141.9	0.0
9		137.3	137.3	0.0
10		131.4	131.4	0.0
11		37.6	38.0	-0.39
12		32.8	33.33	-0.51
13		24.7	25.1	-0.40
14		32.8	33.29	-0.45
15		35.3	35.6	-0.28
16		68.5	68.3	0.19
17		16.3	16.6	-0.25
18		18.6	18.7	-0.14
19		173.7	173.8	-0.09
20		20.7	20.7	0.11

31 of 33

E. Resinosa is named after the thick resin it exudes
Not previously investigated for phytochemicals
Large tubercles present on the stem

5. Eremophila Resinosa

Unpublished results

32 of 33

33 of 33

Acknowledgements

Dr Nicholas Sadgrove

Mr Julian Klepp

Ms Sarah Legendre

Mr Dane Lyddiard

Prof Chris Sumby (UofA)

Prof Vicky Avery (Griffith)

Arid Lands Botanical Gardens

The University of New England for funding