Method for preparing dicaffeoylquinic acids and use thereof in combating aphids
Method for preparing 3,5-dicaffeoylquinic acid and certain derivatives thereof, and use thereof in the preparation of plant-protection products. Method for combating aphids using 3,5-dicaffeoylquinic acid and certain derivatives thereof.
1. A method for preparing compounds of Formula (I)
wherein
R represents a hydrogen atom or a methyl group,
R 1 , R 2 , and R 4 each represent, independently of each other, a hydrogen atom or a caffeoyl group,
R 3 represents a hydrogen, a caffeoyl group, or a succinyl group,
provided that at least two of R 1 through R 4 represent a caffeoyl group, and that
R 3 represents a succinyl group only if R 2 and R 4 represent a caffeoyl group, from nontuberized roots of plants of the genus Ipomoea comprising the following steps:
a) taking samples of said nontuberized roots or recovering root exudate thereof,
b) extraction of phenolic compounds from said roots or said root exudate using one or several organic solvents, and
c) recovery of the raw extract.
2. The method according to claim 1 further comprising the step:
d) purification of said extract obtained in Step c).
3. The method according to claim 1 further comprising the step:
e) spontaneous isomerization of said extracts under alkaline pH conditions; said Formula (I) compounds may be in the form of regio- or stereoisomers, or mixtures thereof.
4. The method according to claim 1 , wherein said nontuberized roots originate from plants selected from the group consisting of the sweet potato ( Ipomoea batatas ), morning glory ( Ipomoea purpurea ), water spinach ( Ipomoea aquatica ), oceanblue morning glory ( Ipomoea indica ), and Scarlett O'Hara morning glory ( Ipomoea nil ).
5. The method according to claim 1 , wherein said nontuberized roots originate from Ipomoea batatas.
6. The method according to claim 1 , wherein nontuberized roots are produced by growing in a liquid medium, under light, and under nitrogen deficiency conditions.
7. The method according to claim 1 , wherein the Formula (I) compounds are selected from the group consisting of 3,5-dicaffeoylquinic (3,5-diCQ) acid, cynarin (1,3-diCQ), 1,5-dicaffeoylquinic (1,5-diCQ) acid, 3,4-dicaffeoylquinic (3,4-diCQ) acid, 4,5-dicaffeoylquinic (4,5-diCQ) acid, methylated analogues thereof, triacylated analogues thereof and mixtures thereof.
8. The method according to claim 1 , wherein the Formula (I) compounds are selected from the group consisting of 3,5-dicaffeoylquinic (3,5-diCQ) acid, cynarin (1,3-diCQ), 1,5-dicaffeoylquinic (1,5-diCQ) acid, 3,4-dicaffeoylquinic (3,4-diCQ) acid, 4,5-dicaffeoylquinic (4,5-diCQ) acid, 3,4,5-tricaffeoylquinic (3,4,5-triCQ) acid, methyl 3,5-dicaffeoylquinate, methyl 3,4-dicaffeoylquinate, methyl 4,5-dicaffeoylquinate, 4-succinyl-3,5-dicaffeoylquinic acid and mixtures thereof.
9. The method according to claim 1 comprising the following steps:
a) taking samples of said nontuberized roots originating from tubers, cuttings, seedlings, or layers,
b) freezing said roots sampled in Step a) in liquid nitrogen,
c) freeze-drying said roots frozen in Step b),
d) grinding said freeze-dried roots in liquid nitrogen, then lyophilizing the freeze-dried roots in order to obtain a dry powder,
e) extracting phenolic compounds from the dry powder using an organic solvent in 1 to 4 passes, via cold stirring,
f) rinsing the final residue from Step e) with the same organic solvent as the one used in Step e), then evaporating the solvent present in the extract until an aqueous phase is obtained,
g) optionally, liquid/liquid volume-to-volume extraction of the aqueous phase from Step f) using an apolar solvent, via several successive extractions, after adding a salt and an acid to said aqueous phase, to obtain an organic phase;
h) dry concentration of the aqueous phase obtained in Step f) or of the organic phase obtained in Step g), after adding a drying agent and filtration in order to eliminate residual water, then taking up the dry residue using an organic solvent,
i) separation the organic phase of Step h) using semipreparative reversed-phase HPLC and collection of the fraction containing 3,5-diCQ,
j) concentration of the fraction obtained in Step i) until an aqueous phase is obtained,
k) liquid/liquid volume-to volume extraction of the aqueous phase obtained in Step j) using an apolar solvent via several successive extractions, after adding a salt to said aqueous phase, in order to facilitate 3,5-diCQ extraction into the apolar solvent, and obtain an organic phase;
l) dry concentration of the organic phase of Step k), after adding a drying agent and filtration in order to eliminate residual water, and taking up the dry residue using an organic solvent, and
m) cold precipitation of the compound by adding water in an amount of at least 3 volumes of water for 1 volume of the organic solvent used in the previous step), and freeze-drying of the precipitated compound in order to obtain the compound in the form of a dry powder.
10. The method according to claim 9 , wherein Step g) is carried out by extracting with ethyl acetate or diethylether (volume-to-volume) after adding NaCl or ammonium sulfate and metaphosphoric acid into the aqueous phase and Step k) is performed via extraction, either by ethyl acetate or by diethylether (volume to volume) after NaCl or ammonium sulfate is added into the aqueous phase.
11. The method of claim 1 , wherein the compounds of Formula (I) are selected from different isomeric forms of dicaffeoylquinic acids, triacylated analogues thereof, methylated analogues thereof, 4-succinyl-3,5-dicaffeoylquinic acid, or mixtures thereof.
12. The method of claim 1 , wherein the compounds of Formula (I) are selected from different isomeric forms of 3,5-dicaffeoylquinic (3,5-diCQ) acid, triacylated analogues thereof, methylated analogues thereof or mixtures thereof.
13. The method of claim 1 , wherein the compound of Formula (I) is 3,5-dicaffeoylquinic (3,5-diCQ) acid.
14. The method of claim 1 , wherein the nontuberized roots are produced by layering, cutting or from seedlings.
15. The method of claim 6 wherein said nontuberized roots originate from plants selected from the group consisting of the sweet potato ( Ipomoea batatas ), morning glory ( Ipomoea purpurea ), water spinach ( Ipomoea aquatica ), oceanblue morning glory ( Ipomoea indica ), and Scarlett O'Hara morning glory ( Ipomoea nil ).
16. The method of claim 6 wherein said nontuberized roots originate from Ipomoea batatas.
17. The method of claim 14 wherein said nontuberized roots originate from plants selected from the group consisting of the sweet potato ( Ipomoea batatas ), morning glory ( Ipomoea purpurea ), water spinach ( Ipomoea aquatica ), oceanblue morning glory ( Ipomoea indica ), and Scarlett O'Hara morning glory ( Ipomoea nil ).
18. The method of claim 14 wherein said nontuberized roots originate from Ipomoea batatas.
19. The method of claim 9 wherein all of steps a) through m) are performed at a temperature ranging from 3 to 5° C., protected from prolonged light exposure, and the acid pH of the aqueous solvents ranges from 5.0 to 6.0.