CharacterizationCharacterization andand valorizationvalorization ofofPolynesianPolynesian plant ressources: plant ressources: tamanu, tamanu, metuapuametuapua’’aa andand puahipuahi
P. Raharivelomanana, T. Teai, S. Soulet, A. Gicquel and J.P. Bianchini
Laboratoire Biodiversité Terrestre et MarineUniversité de la Polynésie Française
PSI 2009
Outlines
Introduction
Tamanu (Calophyllum inophyllum)
Metuapua’a (Microsorum scolopendria, Microsorum membranifolium)
Puahi (Santalum insulare)
Conclusion
Introduction
Tamanu (Calophyllum inophyllum)
Metuapua’a (Microsorum scolopendria, Microsorum membranifolium)
Puahi (Santalum insulare)
Conclusion
Plants uses :Food
MedicinesCosmetics
Secondary metabolites:Alkaloids, polyphenols, saponosides, terpens….
Bioactive Molecules:Medicines directly from natural origin: 40%Synthetic molecules derived from natural products: 20%
Traditional usesTraditional uses Modern usesModern uses
Medicinal plantsUseful plants
Medicinal plantsUseful plants
NutraceuticalsMedicines
Cosmetics….
NutraceuticalsMedicines
Cosmetics….
South Pacific3521 km2 (emerged area) and 5 Millions km 2 of ocean118 islands in 5 archipelagoes
Various ecological conditions :Marquesas - Australes: (7°-28°) South LatitudeAltitude: 0 - 2241m (atolls, islands)Climates, Vegetations, Soils (coral, volcanic)
Original flora:Vascular Plants (Angiosperms et Pteridophyta):
898 indigenous species, 570 endemic species (63%)150 species considered as medicinal plants
Non vascular Plants (Bryophytes, lichens…): poorly investigated
Data analysisBioassays
Bibliography
Sample Collection, botanical identification
Valorizations
PhytochemicalPhytochemical investigationsinvestigations
Compound E.carotovora S. aureus C. accolans C. albicans IC50 (µg/ml) XOD inihibition
Mallotinic acid 13 9 9 9 > 100 Corilagin 20 12 12 12 72.9 Mallorepanin 15 10 10 10 > 100 Chebulagic acid 19 11 10 12 46.3 Ellagic acid 15 0 12 10 2.8 Ellagic acid-4-O-β-D-xylopyranoside
12 0 0 0 2.1
Gallic acid 13 7 7 7 7.1
purificationpurificationpurificationpurification
ssssééééparationsparationsparationsparations
extractionextractionextractionextraction
ppm (f2)1.001.502.00
0.50
1.00
1.50
2.00
2.50
ppm (f1)
NMR
IR
ExtractionIsolations, purifications
Characterizations
collectecollectecollectecollecte
characterizationscharacterizationscharacterizationscharacterizations
Tamanu
Order : Malpighiales
Family : Clusiaceae
Genus : Calophyllum
Species : inophyllum
Vernacular names : Tamanu, Alexandrian Laurel
TaxonomyTaxonomy
Flower
Nuts
Leaf
Bark
EthnobotanicalEthnobotanical uses of Tamanu in uses of Tamanu in PolynesiaPolynesia
� Nuts:– Oil :analgaesic– Wound healing
� Tamanu: � Sacred tree of « marae » surroundings� Precious wood for carvings
� Leaves:– Macerates: treatment of skin diseases (dermatoses, urt icaires, eczemas)– Antidiarrhoic, Antifever– Treatment of conjunctivis– Analgesic: treatment of headache and hemorroids
� Bark:– Macerates: purgative, treatment of ulcers…
NeoflavonoidsNeoflavonoids
O
O
O O
R1
R2
R3O
O
R1
R2
O
OH
R1
R2
O O
O
O
O
O
OOMeO
O
O O
O
O
OH
O
OO
OH
O
Neoflavonoids
Phenyl coumarins
Chromanol
Chromanone
Inophyllum P Inophyllum C Inophyllum G1 Calophyllolid
Plant material : dried leaves (2kg) EtOAc extract (140g)
Flash chromatography
1 23 4
5 6
Isolation and purification of components
Preparatives HPLC RI, DDL or UV-VWD
Purifications on TLC
Structural elucidationsNMR 1H, 13C (1D, 2D),
MS/MS
O O
O
O
OH
O O
O
O
OH
O O
O
O
O
O OH
O
O
COOH
O
OO
OH
O
O
OO
OH
O
10,11-trans stereochemistryin C. inophyllumgrown in French Polynesia
10,11-cis stereochemistrynot detected in C. inophyllum grown in French Polynesia
InophyllumG1
InophyllumG2
Inophyllum A Inophyllum D
Inophyllum EAcide
isocalophyllique
ChemodiversityChemodiversity ofof C. inophyllumC. inophyllum ((leavesleaves, , nutsnuts))
O O
O
O
OH
O O
O
O
OH
O O
O
O
O
O OH
O
O
COOH
O
OO
OH
O
Inophyllum B Inophyllum P
Inophyllum C Acide calophyllique
Inophyllum F
Biological activities of Tamanu constituents (leaves,nuts)
Biological properties Constituents
Antiviral (anti-HIV) Inophyllums B and C…Anticancer Néoflavonoïdes, Calophyllolide…
Anti-inflammatory Phénylcoumarines, Calophyllolide…Antibacterial Inophyllums C et E, Calophyllolide…
Piscicidal Inophyllums C and E…
Others properties: cicatrisation, pain relief, antioxidant…..Others properties: cicatrisation, pain relief, antioxidant…..
Nuts Oil
Cosmeticproducts
Refineryprocess
RefinedOil
HighlyValorizable products
Valorizations of Tamanu oil
Valorization:Short term !!!Valorization:Short term !!!
Metuapua’a
metuapua’a
�Bioactive metabolites: Ecdysteroids
�Medicinal fern in Pacific region and French Polynesia.
Microsorum scolopendria
Microsorum membranifolium
Crushing the plants, extraction with green coconutwater content
2 rhizomes of metua pua'a2 rhizomes of tiatiamoua1 green coconut
Anti-vomitive.Raau Pihae
Crushing the plants, water extraction, and filtration.
Tiare flowerbudsmetua pua'a
General TonicRaau Fati
Crushing the plants and extract withwater
metua pua'aSugar cane
Purgative for children below3 years.
Raau Tahee
PréparationMajor Ingredients
TheurapeuticIndications
RemedyName
Polynesian traditional medecine uses of « metuapua’a »
Metuapua’a Fronds of Metuapua’a
Rhizomes of Metuapua’a and flowerbuds of Tiare
Formulation
Crushing Extraction Filtration
Raau Fatii preparation
Traditional preparation of Metuapua’a potion (M. scolopendria and M. membranifolium)
O
R1
H
OH
R2
R3
CH3
CH3OH
OH
1 9
12
15
19
18
4 6
20
22
A B
C D
2
3 5 7
11
810
14
1316
17
R1=R2=R3=OH=H
Ecdysteroids
� Phytoecdysteroid firstly found in Podocarpus nakaii in 1966 (K. Nakanishi)
� Esters conjugates (cinnamate, coumarate)
� Higher concentration in plants than in animals
� Firstly found in silkworm pupae in 1954
� Polyhydroxylated steroids found both in animals and plants.
� More than 300 structures in more than 3000 species
� Glycosides conjugates (Glu, Gal, Xyl…)
λλλλmax = 245 nm
Bioactivity of Ecdysteroids
Hormone of Regulation of metamorphosis (moulting, ecdysis)Hormone of Regulation of metamorphosis (moulting, ecdysis)
ArthropodsArthropods
MammalsMammalsIncrease of protein synthesis
Immune stimulant
Anti inflammation and pain relief
Wound healing
Antiarrythmic
Hypoglycemic
Hypocholesterolemic
Hepatoprotective
Antidepressant
General tonic (physical, mental, sexual)
Adaptogenic
….
Increase of protein synthesis
Immune stimulant
Anti inflammation and pain relief
Wound healing
Antiarrythmic
Hypoglycemic
Hypocholesterolemic
Hepatoprotective
Antidepressant
General tonic (physical, mental, sexual)
Adaptogenic
….
HPLC LPLC polyamide gel
Plant material
BuOHPhase
Aqueous Phase
H2O:BuOH
Extract
Lipidic Phase
Aqueous Phase
MeOH, 48h MeOH:CHCl3:H2O
EcdysteroidsSpectroscopy: NMR, MS, IR , UV
Fractionation, Isolation and Structure elucidation of Microsorum spp. extracts
O
OH
OHH
CH3
CH3
OH
OH
OH
OH
1
21
9
12
15
19
1820
27
24
4
6
20-hydroxyecdysone (20E)
O
OH
OHH
CH3
CH3
OH
OH
OH
1
21
9
12
15
19
1820
27
24
4
6
Ecdysone (E)
O
OH
OHH
OH
OHOH
O
H
H
H
O1
21
9
12
15
19
1820
4
6
24,28-diépicyastérone O
OH
OHH
OH
O
1 9
12
15
19
1820
4
6
Poststérone
O
OH
OHH
CH3
OH
OH
OH
OHR1
1
21
9
12
15
19
1820
27
24
4
6R1= H inokostérone
R1= CH2-CH3
amarastérone A
O
OH
OHH
CH3
CH3
OH
OH
OH
OHR
1
21
9
12
15
19
1820
27
24
4
6
R= CH2-CH3
makistérone C
R= CH3
makistérone A
Major Ecdysteroids in M. scolopendria extracts
O
OH
OHH
CH3
CH3
OH
OH
OH
OH
1
21
9
12
15
19
1820
27
24
4
6
20-hydroxyecdysone (20E)
O
OH
OHH
C H 3
C H3
O H
O H
O H
1
21
9
12
15
19
1820
27
24
4
6
Ecdysone (E)
O
OHH
OH
OH
OH
OH
1
21
9
12
15
19
1820
4
6
2-deoxy-20-hydroxyecdysone (2d20E)
Major Ecdysteroids in M. membranifolium extracts
O
O H
OH
OH
HOH
2-deoxyecdysone (2dE)
R.Ho, T. Teai, D. Loquet, J.P. Bianchini, J.P. Girault, R. Lafont and P. Raharivelomanana, Nat. Prod. Com. , 2 (3): 239-242-8 (2007)
O
OH
OHH
CH3
CH3
OH
OH
OH
OHR
1
21
9
12
15
19
1820
27
24
4
6
R= CH2-CH3
makistérone C
R= CH3
makistérone A O
OH
OHH
CH3
OH
OH
OH
OHR1
1
21
9
12
15
19
1820
27
24
4
6R1= H inokostérone
R1= CH2-CH3
amarastérone A
New ecdysteroids conjugates in M. membranifolium extracts
Rhamnoside conjugate (compound 1)
MS (CI/D) ions at m/z:
612 (M + NH4)+, 594 (MH)+, 594 (M)+
577 (MH – H2O)+, 559 (MH –2 H2O)+
448 (M + NH4 - sugar)+, 431 (MH - sugar)+,
413 (MH – sugar - H2O)+, 395 (MH – sugar - 2H2O)+
164 (sugar)+ : deoxy-hexose
λλλλmax = 245 nm
1H -13C NMR data (C5D5N + εεεε D20) :close similarity to 2dE
modification of the side chain
sugar linked to C-25
sugar moiety = Rhamnose
UV MS NMR
Compound 1 : 2-deoxyecdysone 25-α- L-rhamnopyranoside
R.Ho, J.P. Girault, P.Y Cousteau, J.P. Bianchini, P. Raharivelomanana, R. Lafont, J. Chrom. Sci, 46: 1-8 (2008)
Ferulates conjugates (compounds 2 and 3 )
O
OH
OH
OH
HO
O
O
O
OH
OH
OH
OH
R
O
(R = OH) Compound 2 : 2-deoxy 20-hydroxyecdysone 3-[-4 (1-ββββ-D glucopyranosyl)]-ferulate
(R = H) Compound 3 : 2-deoxyecdysone 3-[-4 (1-ββββ-D glucopyranosyl)]-ferulate
New class of Glucosyl-ferulates ecdysteroids
R.Ho, J.P. Girault, P.Y Cousteau, J.P. Bianchini, P. Raharivelomanana, R. Lafont, J. Chrom. Sci, 46: 1-8 (2008)
Chemical composition of M.scolopendriaand M. membranifoliumextracts
0.240.12n.d0.12Makisterone A
0.090.500.407.072-deoxy-20-hydroxyecdysone
3.411.450.874.91Ecdysone
0.420.360.020.2Inokosterone
11.174.692.0216.63Concentration (mg.g -1)
0.25n.d0.090.48Makisterone C
6.762.230.642.1320-hydroxyecdysone
RhizomeFrondRhizomeFrondPart
M. scolopendriaM. membranifoliumSpecies
* M. scolopendria: higher concentration in rhizomes* M. membranifolium: higher concentration in fronds.
R.Ho, T. Teai, D. Loquet, J.P. Bianchini, J.P. Girault, R. Lafont and P. Raharivelomanana, Nat. Prod. Com. , 2 (3): 239-242-8 (2007)
Good candidates for nutraceuticals compounds !!!
Solubility in H2O and different solvents
Metabolic stability
Ability to cross membrane
Bioavailability (ADME): Absorption,
Distribution, Metabolism, Excretion
Extremely low toxicity (DL50: 6 g.kg-1)
Solubility in H2O and different solvents
Metabolic stability
Ability to cross membrane
Bioavailability (ADME): Absorption,
Distribution, Metabolism, Excretion
Extremely low toxicity (DL50: 6 g.kg-1)
Physical, Chemical and Biological CharacteristicsPhysical, Chemical and Biological Characteristics
Characteristics of Ecdysteroids
Formulations
Dosage forms (capsule, beverage, tablets, powder, pills, alcoholic extracts…)
Association with other preparations (Green tea, Soups, Drinks…)
….
Formulations
Dosage forms (capsule, beverage, tablets, powder, pills, alcoholic extracts…)
Association with other preparations (Green tea, Soups, Drinks…)
….
Complementary data !!!
Metuapua’a valorization potentials
* anabolic
* sexual tonic
* anti-depressant
* Slimming
Valorization:Mid-term !!!Valorization:Mid-term !!!
Sandalwood
Biogeographical distribtion of sandalwood
carvings
Incense
Perfumes
Uses of sandalwood in the world
Overexploitation since the 19th century
Santalum insulare : ahi, puahi, …
Morphology
Uses of sandalwood in Polynesia
Carvings Traditional medicine Monoï
Isolation of sesquiterpenoids of S. insulare
Dried and chopped
Fractionation and isolation
LPLC (SiO 2)
NP- HPLC
Extraction (n-hexane)
SpectroscopyNMR, MSsesquiterpenoids
5
10
79
61
12
15
13
8
2
14
11
4
3
CH3
CH2OH
OHOH
HaHb
1110
15
5
7
14
13
212
3
9
8
6
4
1
CH2OH
10
14
15
13
11
12
9
7
8
6
3
12
54
HO
OH
5
10
7
96
1
12
15
13
82
14
11
43
CH3HOH2C
HHO
Distillation
Butaud et al . 2003. J. of Ess. Oil Res., 15: 323-326.
Chemotypes of S.insulare (GC)
Individus (axes F1 et F2 : 48.00 %)
-4
-2
0
2
4
6
8
-6 -4 -2 0 2 4
-- axe F1 (29.59 %) -->
NH crêtes de Toovii
NH Haut de TD
NH Toovii
NH Taiohae-Vaioa
NH Vaiteheihi
NH Motuhee
NH Maauu
Ua Pou
HO Hanaavao
HO Taaoa
HO Hanauaua
HO Intérieur
Tahuata
Fatu Hiva
(Z)-nuciferol chemotype
Santalols chemotype
Chemiodiversity of Santalum insulare from essential oil content
PCA of sandalwood extracts from Marquesas islands
Santalol content of Santalum species
0 - 191 - 49S. lanceolatum
16 - 2441 - 55S. album* ( ISO)
18 - 2346 - 56S. insulare
13 - 3424 - 57S. yasi
2 - 261 - 54S. austrocaledonicum
0 - 90 - 22S. spicatum
0 - 240 - 47S. macgregorii
β-santalolα-santalol
*Huile essentielle de S. album > 2000 Euros/lkg
Flavonoids of Santalum insulare leaves
O
OH
R'
O
R'''
R''O
R''''
OH
Name R’ R’’ R’’’ R’’’’
api-7-neo a H neohespc H H
lut-7-neob OH neohespc H H
isovitexin H H H β-D-gluc d
vitexin H H β-D-gluc d H
isoorientin OH H H β-D-gluc d
vicenin-2 H H β-D-gluc d β-D-gluc d
lucenin-2 OH H β-D-gluc d β-D-gluc d
a apigenin-7-O-neohesperidoside.b luteolin-7-O-neohesperidoside.
Butaud et al (2006)
O-Glycosylflavones
C-Glycosylflavones
O
OH
R'
O
R'''
R''O
R''''
OHa apigenin-7-O-neohesperidoside.b luteolin-7-O-neohesperidoside.
Name min max mean s. d.api-7-neo a 0.1 15.0 1.1 2.2lut-7-neob 1.3 49.6 14.7 8.2isovitexin 0.1 25.4 7.1 6.1
vitexinisoorientin
0.7 13.9 2.6 2.12.0 50.0 28.4 13.6
vicenin-2 14.9 78.7 37.2 15.7lucenin-2 0.6 33.3 8.8 7.7
Flavonoid compositions of leaves of Santalum insulare
-6
-4
-2
0
2
4
6
-6 -4 -2 0 2 4 6
PC 1
Variety
insulare
alticola
raiateense
marchionense
raivavense
margaretae
I-vit
+
Lut-7-neo +
+Vit
+ Api-7-neo
+ Luc-2+ Vic-2+I-ori
PC 2
Society archipelago
Austral archipelagoMarquesas archipelago
-6
-4
-2
0
2
4
6
-6 -4 -2 0 2 4 6
PC 1
Variety
insulare
alticola
raiateense
marchionense
raivavense
margaretae
I-vit
+
Lut-7-neo +
+Vit
+ Api-7-neo
+ Luc-2+ Vic-2+I-ori
PC 2
Society archipelago
Austral archipelagoMarquesas archipelago
Chemodiversity of Santalum insulare from flavonoid content
Restoration and conservation
Valorization:Long term !!!Valorization:Long term !!!
ConclusionsConclusionsConclusions
- Perfume- CosméticsLong term
PuahiS. insulare
- Nutraceutical- Cosmetical
- Pharmaceutical…..
Mid termMetuapua’a
M. scolopendriaM. membranifolium
- Cosmetic- PharmaceuticalShort term
TamanuC. inophyllum
DomainValorizationPlant
Maururu!!!PSI organization
Fiu…Encore des plantes!!
Laboratoire Biodiversité Terrestre et Marine EA 4239