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