Process of obtaining thylakoids from photosynthetic organisms; plant fractions obtained from the process; pure thylakoids; and methods of use of thylakoids as ROS scavengers, photo-protectors, biosensors, biofilters and bioreactors
View Patent ↗This invention relates to a process by which an extract comprising integral thylakoids is obtained. The resulting extract is a potent dynamic antioxidant useful as a ROS (reactive oxygen species) scavenger. This extract is intended to be used for the treatment or prevention of diseases involving the generation of ROS, such as inflammatory diseases or cancer. This extract also finds a use as a solar screen because of its capacity to capture UV radiations and to dissipate the solar energy into heat.
1. A method of obtaining an extract from a photosynthetic organism, said method comprising the steps of:
a) providing a suspension of organism constituents that contain thylakoids;
b) disrupting the constituents while recovering thylakoids under light conditions which minimize light flux, in a medium having a viscosity comprised between 1 to 1.3 centipoise and a pH above 2 and below 10; the medium being added in a volume calculated upon the following equation:
(
Volume
of
medium
+
organism
constituents
water
content
)
(
Organism
constituents
dry
weight
)
>
10
whereby a first extract essentially constituted of thylakoids, cells debris/membranes, and a liquid phase is obtained, said thylakoids comprising organized photosynthetic pigments;
c) separating thylakoids, cell debris/membranes and liquid phase from each another, to form a second, third and fourth extracts essentially constituted by isolated thylakoids, cell debris/membranes, and a liquid phase, respectively; and
d) eliminating any electron donor from said first, second and third extracts.
2. The method according to claim 1 , wherein the equation is:
(
Volume
of
medium
+
organism
constituents
water
content
)
(
Organism
constituents
dry
weight
)
=
25
-
150.
3. The method according to claim 1 , wherein the pH is between 5 and 8.
4. The method according to claim 1 , wherein the pH is between 6 and 7.5.
5. The method according to any one of claims 1 , 2 , and 4 , wherein said organism is a plant.
6. The method according to claim 5 , wherein the suspension of step a) is obtained by mechanically dispersing plant constituents or tissues in said medium.
7. The method according to claim 5 , wherein step a) is preceded by a step of submitting a plant to a conditioning parameter selected from light, osmotic stress, heat, cold, freezing, dryness, hormones, chemical and biological inducers.
8. The method according to claim 1 , wherein step a) is preceded by a step of conditioning said organism in a light environment of a wavelength comprised between about 500 and 600 nm, and step b) is performed under the same light conditions.
9. The method according to claim 5 , wherein step a) is preceded by a step of conditioning said plant in a light environment of a wavelength comprised between about 500 and 600 nm, and step b) is performed under the same light conditions.
10. The method according to claim 1 , wherein said viscosity is partly achieved by adding a sugar in the medium.
11. The method according to claim 1 , wherein said viscosity is partly achieved by the presence of sorbitol in a concentration of about 0.2 to 1.5 M in said medium or of a sugar achieving a viscosity equivalent to 0.2 to 1.5 M sorbitol.
12. The method according to claim 1 , wherein said viscosity is partly achieved by the presence of sorbitol in a concentration of about 0.2 to 0.4 M in said medium or of a sugar achieving a viscosity equivalent to 0.2 to 0.4 M sorbitol.
13. The method according to any one of claims 1 to 4 , and 6 to 12 , wherein said medium has the following composition: Tris or acetate or ascorbate buffer (20 mM) having a pH of 7.5 and sorbitol or sucrose or fructose 350 mM.
14. The method according to any one of claims 1 to 4 , and 6 to 12 , wherein the step of separating is performed upon a difference of sedimentation coefficient of each of thylakoids, cell debris and membranes, and liquid phase.
15. The method according to claim 14 , wherein the step of separating comprises centrifuging the fiat extract in a tube equipped with a filter in a superior portion of the tube, the filter having a porosity onto which cell debris and membranes deposit while the thylakoids and the liquid phase pass through the filter, the thylakoids forming a pellet in an inferior portion of the tube.
16. The method according to any one of claims 1 to 4 , 6 to 8 , and 10 to 12 , wherein said electron donor is water.
17. The method according to claim 16 , wherein water is eliminated under vacuum freeze drying.
18. The method according to claim 16 , wherein water is eliminated by exchanging it against an amphoteric solvent or surfactant after step c).
19. The method according to claim 16 , wherein water is eliminated by exchanging it against propylene glycol.
20. The method according to claim 5 , wherein said viscosity is partly achieved by the presence of sorbitol in a concentration of about 0.2 to 0.4 M in said medium or of a sugar achieving a viscosity equivalent to 0.2 to 0.4 M sorbitol.
21. The method according to claim 5 , wherein said medium has the following composition: Tris or acetate or ascorbate buffer (20 mM) having a pH of 7.5 and sorbitol or sucrose or fructose 350 mM.
22. The method according to claim 5 , wherein said electron donor is water.
23. The method according to claim 9 , wherein said electron donor is water.
24. The method according to claim 22 , wherein water is eliminated under vacuum freeze drying.
25. The method according to claim 23 , wherein water is eliminated under vacuum freeze drying.
26. The method according to claim 22 , wherein water is eliminated by exchanging it against propylene glycol.
27. The method according to claim 23 , wherein water is eliminated by exchanging it against propylene glycol.
28. A method of obtaining an extract from a plant, said method comprising to steps of:
a) exposing the plant to a light environment of a wavelength comprised between about 500 and 600 nm,
b) providing a suspension of plant constituents that contain thylakoids;
c) disrupting the constituents while recovering thylakoids under the light environment of step a), in a medium having a viscosity comprised between 1 to 1.3 centipoise and comprising Tris or acetate or ascorbate buffer (20 mM) having a pH of 7.5, and sorbitol or sucrose or fructose 350 mM; the medium being added in a volume calculated upon the following equation:
(
Volume
of
medium
+
organism
constituents
water
content
)
(
Organism
constituents
dry
weight
)
=
25
-
150.
whereby a first extract essentially constituted of thylakoids, cells debris/membranes, and a liquid phase is obtained, said thylakoids comprising organized photosynthetic pigments;
d) separating thylakoids, cell debris/membranes and liquid phase from each another, to form a second, third and fourth extracts essentially constituted by isolated thylakoids, cell debris/membranes, and a liquid phase, respectively; and
e) eliminating any electron donor from said first, second and third extracts.