Field of the invention
The present invention relates to the filed of extraction of xanthophylls from
respective oleoresin source. More particularly, the present invention relates to the
filed of extraction of carotenoid stereoisomers from marigold oleoresin, particularly
extraction of different stereo-isomeric molecules of the carotenoids, viz. lutein, (3R,
3’S) meso-zeaxanthin and (3R, 3’R) trans-zeaxanthin from the marigold oleoresin,
although not exhaustive of the source of such oleoresin.
Background of the Invention
Xanthophylls are the carotenoids responsible for the pigmentation found as well as
those which are naturally synthesized in plants, algae, including certain bacteria and
fungi. These are such pigmented compounds which are responsible for the color
thereof the respective fruit or vegetable or the flower. Marigold flower is one such
flower in which a widely known xanthophyll called as lutein is abundant, wherein
the said lutein in marigold is present in the ester form of various fatty acids such as
stearic acid, palmitic acid etc. These lutein ester(s) are responsible for the
characteristic yellow color of the marigold flower apart from also being helpful to
protect the plants against external threats such as ultra-violet light, and pathogenic
invasions in the plant. Whereas plants can synthesize lutein, humans or other
animals are devoid of synthesizing lutein. However, lutein is concentrated in the
macular region of the human eye which is a very important constituent for eyehealth altogether, to be obtained from food, thus being an essential dietary
ingredient. The macula lutea (i.e. the yellow spot) in the retina contains lutein in three variants of its isomeric forms lutein, meso-zeaxanthin, and zeaxanthin.
Zeaxanthin can exist in three different stereo-isomeric forms in nature, namely
zeaxanthin (the 3R, 3'R form), meso-zeaxanthin (the 3R, 3'S form) and (3S, 3'S)
zeaxanthin. The least naturally available stereo-isomer of lutein is Mesozeaxanthin.
Whereas there is suitably satisfying evidence of natural occurrence of mesozeaxanthin from animal sources like Mexican hen egg yolk, some species of fishes,
shrimp, and sea turtle (Rasmussen H, Muzhingi T, Eggert T, Johnson EJ. Lutein,
zeaxanthin, meso-zeaxanthin content in egg yolk and their absence in fish and seafood. J Food Composition Anal 2012; 27: 139–144) but the said isomeric form
is not reportedly available from plant sources. Meso-zeaxanthin was first isolated
from shrimps and turtles by Moaka et. Al. (Maoka T, Arai A, Shimizu M, Matsuno
T. The first isolation of enantiomeric and meso-zeaxanthin in nature. Comp
Biochem Physiol B 1986; 83: 121–124). The said study hypothecated the
biocatalytical degradation or conversion to meso-zeaxanthin from lutein. Yet, a
subsequent study by J.M. Nolan et al. negated the possibility of automatic
conversion into meso-zeaxanthin from lutein within the biological system stating
that, saponification of purified lutein (L) does not generate mesozeaxanthin (MZ),
even with high concentrations of KOH. (Nolan JM, Meagher K, Kashani S, Beatty
S. What is meso-zeaxanthin, and where does it come from? Eye (Lond). 2013
Aug;27(8):899-905. doi: 10.1038/eye.2013.98. Epub 2013 May 24. PMID:
23703634; PMCID: PMC3740325). Further, meso-zeaxanthin is found to be
enzymatically converted from other two isomeric forms of lutein in the human eye
(Bernstein PS, Li B, Vachali PP, Gorusupudi A, Shyam R, Henriksen BS, Nolan
JM. Lutein, zeaxanthin, and meso-zeaxanthin: The basic and clinical science
underlying carotenoid-based nutritional interventions against ocular disease. Prog
Retin Eye Res. 2016 Jan;50:34-66. doi: 10.1016/j.preteyeres.2015.10.003. Epub
2015 Nov 2. PMID: 26541886; PMCID: PMC4698241). Meso-zeaxanthin (the 3R,
3'S form) is found in the epicenter of the macula, (3R,3´R)-zeaxanthin in the midperipheral macula and lutein at the peripheral macula are the main carotenoids in
the macula lutea, found in a ratio of 1:1:1, and are collectively referred to as macular
pigment (MP). Whereas the stereo-isomer meso-zeaxanthin is abundant in the
central macular region, lutein occupies most of the peripheral region in almost
equivalent quantities of meso-zeaxanthin. Additionally, zeaxanthin (3R, 3’R form)
is scattered in between the presence of the other two forms of stereoisomer. (Bone,
R. A.; Landrum, J. T.; Friedes, L. M.; Gomez, C. M.; Kilburn, M. D.; Menendez,
E.; Vidal, I.; Wang, W. (1997-02-01). "Distribution of lutein and zeaxanthin stereoisomers in the human retina". Experimental Eye Research. 64 (2): 211–218.).
An adequate macular pigment optical density (MPOD) is known to aid and retain
proper ocular health, by polarizing or ionizing the direct ultraviolet or blue light falling on the eyes, and/or further protecting the eye against different infections,
and also known in the reduction of age-related macular degeneration (ARMD) in
the eye. Therefore, a number of dietary supplements containing lutein or isomeric
admixtures of lutein, meso-zeaxanthin and zeaxanthin are commercially available
as on date. The most easy and abundant source of lutein extraction for commercial
purposes is marigold flower that has more than 95% content of lutein ester.
However, as enumerated above i.e. lack of meso-zeaxanthin in plant sources, the
said isomeric form is also absent in marigold or sometimes believed to be present
in very very negligible and insignificant amounts. The natural occurrence of Lutein
and Zeaxanthin in Marigold flowers occur approximately in the ratio of 95:5 as dry
weight of the marigold flower.(“Effect of drying treatments on the contents of lutein
and zeaxanthin in orange- and yellow-cultivars of marigold flower and its
application for lutein ester encapsulation”, Materials Science and Engineering 509
(2019) 012060). As regards the extent of research in this particular domain, separate
and specific data on the amount of various isomeric forms of zeaxanthin is missing
since most research conducted in this area of identifying zeaxanthin content in
marigold (or any other source) has endeavoured only with respect to finding the
total zeaxanthin content only and not focussed in calculating the different isomeric
forms of zeaxanthin available in the marigold flower. Yet, it is obvious that the
amount of mesozeaxanthin if at all ought to be present in the marigold flower,
should be in very scarce quantities as total zeaxanthin is itself present in up to
approximately maximum of 5% only.
Irrespective of the source or the mechanism of formation thereof, the presence of
meso-zeaxanthin in the human eye has already been acknowledged by technical
fraternity and therefore, certain dietary supplements of different isomeric
combinations of lutein containing lutein, zeaxanthin and meso-zeaxanthin has been
preferred to address various ocular health related deficiency or medical indications
related thereto, over earlier existing dietary supplements which contained only
lutein and zeaxanthin as the active ingredients. A very early disclosure may be
found in WO1996040092 wherein an admixture of different isomeric forms of
lutein (either with lutein and mesozeaxanthin, or alternatively only lutein as the active ingredient) along with an antioxidant has been claimed. An oral supplement
containing only mesozeaxanthin between 10 mg to 50 mg per day has been to be
administered for increasing the serum carotenoid concentration to at least 0.5 μg/ml
and maintain the increased serum carotenoid concentration at or above 0.5 μg/ml
for at least 14 days, and at least until the macular concentration of carotenoid(s) has
achieved equilibrium is provided in US6218436B1. However, lack of knowledge
in extracting specifically mesozeaxanthin from the marigold flower in the current
state of the art renders such desirable combination of all the three isomeric forms
highly inaccessible and hence their availability is very much limited.
There are some reported patents which disclose methods of production of mesozeaxanthin. US552349A discloses a process to isomerize lutein into zeaxanthin
from marigold oleoresin using strong aqueous oversaturated alkaline solution
(elected from the group consisting of potassium hydroxide, sodium hydroxide,
sodium carbonate, ammonium hydroxide, ammonia, or their mixtures, or in its
anhydrous form or alkali in which the alkali is an organic base selected from the
group consisting of ethanolamine, ethylamine, morpholine, or mixtures thereof).
Similarly, another process is disclosed in US5780693A for manufacturing
zeaxanthin from lutein in presence of solvents such as hexane, heptane, petroleum
ether, or dimethyl sulphoxide, and/or liquid aliphatic or aromatic carbon along with
an alkali hydroxide at given ratios in presence of a phase transfer catalyst.
US6262284B1 mentions a process for simultaneously extracting, saponifying (with
a base NaOH or KOH at pH 12, for 2 hours), and isolating lutein and zeaxanthin,
and a mixture of several rare carotenoids in high purity from plants without the use
of harmful organic solvents. However, this patent does not disclose a simultaneous
method of extracting all the three desired isomeric combinations of lutein,
zeaxanthin and mesozeaxanthin. Another disclosure CN109232345 provides a
method for extracting and isolating lutein crystals from vegetable oils containing
lutein diester, wherein an enzyme solution with an alcohol extract containing the
lutein ester is prepared and filtered to obtain a crystal cake, and then treating the
crystal cake with non-polar organic solvents (selected from ethyl acetate, iso-butyl

Claims:
We claim:
1. A method of extraction of an isomeric combination of (3R,3R,6R) lutein, (3R,
3’S) meso-zeaxanthin and (3R, 3’R) trans-zeaxanthin, the said method comprising
the steps of:
(a) obtaining of marigold oleoresin from dehydrated marigold flowers, wherein
dehydrated marigold [ratio of dehydrated marigold to extraction solvent is
1(dehydrated marigold):10 (extraction solvent)] flowers are treated with an
extraction solvent, wherein the said extraction solvent is selected from ethylene
dichloride, or an unique combination mixture of extraction solvent consisting of
hexane and ethylene dichloride each mixed at a ratio of 1:1 at a temperature range
between 40oC to 70oC under constant rotation for a time span of 8 to 10 hours to
form an extract, which is then evaporated to obtain a thick marigold oleoresin;
(b) treating the oleoresin so obtained in the previous step (a) with isopropyl alcohol
at an amount of three times the weight of oleoresin taken, and then subsequently
with water at a concentration range of 5% the weight of oleoresin taken, thereafter
with an alkali mixture along with a catalyst combination both together (i.e. the alkali
mixture and the catalyst mixture) taken at an amount equivalent to the weight of
oleoresin taken for a period between 8 to 10 hours at a temperature between 100oC110oC under nitrogen blanket to maintain anaerobic conditions to avoid degradation of the marigold oleoresin; wherein the above-mentioned reaction mixture containing the marigold oleoresin,
isopropyl alcohol, water, alkali mixture and the catalyst is being analysed under
high performance liquid chromatography after each 2-hour intervals till the area%
of free (3R,3R,6R) lutein is equal to the area % (3R, 3’S) meso-zeaxanthin is
achieved;
(c) the reaction mixture of step (b) is neutralized by treating with 20% by weight of
citric acid added with water equivalent to the six times the initial weight of oleoresin
taken at the initial step (a) until the pH is brought at 7 within a period of 1 hour at
60oC, and then cooled to room temperature followed by filtering the said reaction
mass through filter press of 5-micron mesh size, and the residue is collected in the
form of a wet cake;
(d) washing the wet cake of step (c) above with hot water, wherein five times the
weight of the said wet cake of hot water is used, and this particular step of washing
the wet cake is undergone for at least 4 times, thereafter filtered through Nutsche
filter to obtain the residue;
(e) the residue from the above step (d) is then subjected to centrifugation at 1400
rotations per minute for 1 hour, to obtain a wet mass;
(f) the said wet mass from the above step (e) is transferred to a reactor mixed with
water equivalent to at least four times the weight of the said wet mass to remove
the additional solvent residues, and then stirred for a period of at least 1 hour, and
thereafter spin dried at 1400 rotations per minute till a lump of wet mass is obtained;
(g) the lump of wet mass so obtained in the previous step is then wet milled using
8 mm mesh in multi-mill, followed by fluidized bed drying at temperature less than
60oC to obtain the desired isomeric combination of (3R,3R,6R) lutein, and (3R,
3’S) meso-zeaxanthin and also (3R, 3’R) trans-zeaxanthin, which is thereafter
sieved and checked under magnetic particles separation to avoid any heavy metal
contamination, to obtain at least 99% pure isomeric combination of (3R,3R,6R)
lutein, and (3R, 3’S) meso-zeaxanthin and also (3R, 3’R) trans-zeaxanthin, wherein
the per unit recovery of the said isomeric combination is at least 96% to 99.2%.
2, The method as claimed in claim 1, wherein the said alkali mixture is a
combination of sodium hydroxide present in a concentration of 70% w/w and
calcium hydroxide present in a concentration of 27% w/w in the alkali mixture
along with the catalyst combination both together.
3. The method as claimed in claim 1, wherein the said catalyst mixture is present in
a concentration of 3% wt/wt in the alkali mixture along with the catalyst
combination both together, the said catalyst being a combination of tetrabutyl ammonium hydrogen sulphate, an isothiocyanate and a polysulfide all these three
said specific components of the catalyst mixture present at a ratio of 1:1:1 in terms
of their respective weight(s).
4. The method as claimed in claim 1, wherein the isomeric combination of
(3R,3R,6R) lutein, and (3R, 3’S) meso-zeaxanthin and (3R, 3’R) trans-zeaxanthin
so obtained in claim 1(g) comprises in equal proportion of quantities of (3R,3R,6R)
lutein, and (3R, 3’S) meso-zeaxanthin each at an amount between 10 mg- 11mg,
and the said (3R, 3’R) trans-zeaxanthin is present at a concentration between 2 mg
to 3 mg.
5. An isomeric combination comprising (3R,3R,6R) lutein, and (3R, 3’S) mesozeaxanthin and (3R, 3’R) trans-zeaxanthin, wherein (3R,3R,6R) lutein, and (3R,
3’S) meso-zeaxanthin and (3R, 3’R) each are present at an equal concentration
range between 40% w/w to 50% w/w, and trans-zeaxanthin is present at a
concentration range between 9% w/w to 10% w/w, wherein the said isomeric
combination is prepared by the process comprising the steps of:
(a) obtaining of marigold oleoresin from dehydrated marigold flowers, wherein
dehydrated marigold [ratio of dehydrated marigold to extraction solvent is
1(dehydrated marigold):10 (extraction solvent)] flowers are treated with an
extraction solvent, wherein the said extraction solvent is selected from ethylene
dichloride, or an unique combination mixture of extraction solvent consisting of
hexane and ethylene dichloride each mixed at a ratio of 1:1 at a temperature range
between 40oC to 70oC under constant rotation for a time span of 8 to 10 hours to
form an extract, which is then evaporated to obtain a thick marigold oleoresin;
(b) treating the oleoresin so obtained in the previous step (a) with isopropyl alcohol
at an amount of three times the weight of oleoresin taken, and then subsequently
with water at a concentration range of 5% the weight of oleoresin taken, thereafter
with an alkali mixture along with a catalyst combination both together (i.e. the alkali
mixture and the catalyst mixture) taken at an amount equivalent to the weight of
oleoresin taken for a period between 8 to 10 hours at a temperature between 100oC 110oC under nitrogen blanket to maintain anaerobic conditions to avoid degradation
of the marigold oleoresin;
wherein the above-mentioned reaction mixture containing the marigold oleoresin,
isopropyl alcohol, water, alkali mixture and the catalyst is being analysed under
high performance liquid chromatography after each 2-hour intervals till the area%
of free (3R,3R,6R) lutein is equal to the area % (3R, 3’S) meso-zeaxanthin is
achieved;
(c) the reaction mixture of step (b) is neutralized by treating with 20% by weight of
citric acid added with water equivalent to the six times the initial weight of oleoresin
taken at the initial step (a) until the pH is brought at 7 within a period of 1 hour at
60oC, and then cooled to room temperature followed by filtering the said reaction
mass through filter press of 5-micron mesh size, and the residue is collected in the
form of a wet cake;
(d) washing the wet cake of step (c) above with hot water, wherein five times the
weight of the said wet cake of hot water is used, and this particular step of washing
the wet cake is undergone for at least 4 times, thereafter filtered through Nutsche
filter to obtain the residue;
(e) the residue from the above step (d) is then subjected to centrifugation at 1400
rotations per minute for 1 hour, to obtain a wet mass;
(f) the said wet mass from the above step (e) is transferred to a reactor mixed with
water equivalent to at least four times the weight of the said wet mass to remove
the additional solvent residues, and then stirred for a period of at least 1 hour, and
thereafter spin dried at 1400 rotations per minute till a lump of wet mass is obtained;
(g) the lump of wet mass so obtained in the previous step is then wet milled using
8 mm mesh in multi-mill, followed by fluidized bed drying at temperature less than
60oC to obtain the desired isomeric combination of (3R,3R,6R) lutein, and (3R,
3’S) meso-zeaxanthin and also (3R, 3’R) trans-zeaxanthin, which is thereafter
sieved and checked under magnetic particles separation to avoid any heavy metal
contamination, to obtain at least 99% pure isomeric combination of (3R,3R,6R) lutein, and (3R, 3’S) meso-zeaxanthin and also (3R, 3’R) trans-zeaxanthin, wherein
the per unit recovery of the said isomeric combination is at least 96% to 99.2%.
6. The isomeric combination as claimed in claim 5, comprising (3R,3R,6R) lutein,
and (3R, 3’S) meso-zeaxanthin and (3R, 3’R) trans-zeaxanthin, wherein
(3R,3R,6R) lutein, and (3R, 3’S) meso-zeaxanthin and (3R, 3’R) each are present
at an equal concentration range between 40% w/w to 50% w/w, and transzeaxanthin is present at a concentration range between 9% w/w to 10% w/w
wherein the stability of the said isomeric combination of (3R,3R,6R) lutein, and
(3R, 3’S) meso-zeaxanthin and also (3R, 3’R) trans-zeaxanthin is comparatively
higher as compared to the stability of the admixture of the said three isomers, or
each of the said isomers present severally, for a period of at least 6 months at
accelerated conditions 40°C ± 2°C and 75%± 5% humidity.