Description:TECHNICAL FIELD OF THE INVENTION:
The present invention relates to a green process for isolation of total marigold extract comprising bioactives rich in lutein ester and flavonoids. The present invention further relates to the process for isolation and purification of bioactives, Lutein and Quercetagetin using green solvents.

BACKGROUND AND PRIOR ART:
Carotenoids are a class of natural fat-soluble pigments found principally in plants, algae, and photosynthetic bacteria, where they play a critical role in the photosynthetic process. Lutein, (β-ε-carotene-3-3-diol) and Zeaxanthin (β-β-carotene-3-3′-diol) are two important carotenoid pigments produced by plants that give fruits and vegetables their yellow to reddish colour. They belong to Xanthophylls group in the carotenoids family with highly reactive hydroxyl groups which cannot be synthesized by humans and animals and it needs to be supplemented via food. Dietary carotenoids are beneficial as antioxidants which protect from diseases and enhance the immune system. Dietary carotenoids sources include a variety of fruits and vegetables, particularly tomato and tomato products. In addition to these, egg yolk is a rich source of lutein and zeaxanthin.

Lutein is the most abundant carotenoid in eye and brain. Lutein in the macula filters blue light, quenches free radicals and supports vision. As a result, lutein may play a role in prevention of age-related eye diseases, such as age-related macular degeneration and age-related cataract. Provitamin A carotenoids are converted into vitamin A, which is essential for growth, immune system function, and eye health.

Marigold flower, along with lutein, contain many other flavonoids and polyphenols which have very high antioxidant activities. Most abundant flavonoids are Quercetagetin, Luteolin, Patuletin. There is enough evidence based scientific literature available for the antioxidant, anti-inflammatory and anti-cancer effect of quercetagetin. Several processes have been used for extracting flavonoids and polyphenols from plant sources.

EP 1371641 A1 discloses a process for the production of Carotenoid crystals from a Carotenoid containing plant oleoresin. Process claims mixing the marigold oleoresin with a water immiscible organic solvent and then saponifying with an alcohol and alkali to a sufficient time for completing the saponification under reflux temperature of the water immiscible organic solvent. Allowing the reaction mixture to separate into organic and aqueous phases and distilling the organic phase to get the Carotenoid.

US 3523138 discloses a process of obtaining xanthophylls from marigold petal meal using hexane, isopropyl ether or chloroform, which is a well-known method of isolating xanthophylls/carotenoids from marigold.

US 6191293 discloses a process of extracting xanthophyll esters from dried marigold corollas using hydrocarbon solvents like pentane, hexane, heptane or mixture of the same disclosed.

CN 110105257B discloses an industrial extraction of Quercetagetin from marigold flowers. The method claims simultaneous extraction of lutein and marigold quercetin using a mixture of n-hexane and acetone. The mixture is separated by two phase extraction using methanol as secondary solvent to get quercetagetin and xanthophylls separated.

CN 102190646A discloses a method for preparing high purity quercetagetin and belongs to the technical field of flavonoid compound extraction. The method comprises the following steps of extracting fermented residue subjected to marigold flower lutein ester extraction by using 5 to 90 percent aqueous solution of ethanol to obtain a crude extract of quercetagetin.

CN 110105257 B discloses an industrial preparation method of quercetagetin, wherein, Marigold residues are uniformly mixed with alcohol, the mixture is heated to the extraction temperature and extracted in an ultrasonic-assisted extraction manner, then, centrifugal filtration, concentration, centrifugal filtration and purification are performed, and finally, a quercetagetin product is obtained.

CN 105693676A discloses a method of separating and purifying quercetagetin from Tagetus erecta. The method includes a step of drying Tagetus erecta dry flowers, grinding into powder, dipping with ethanol or performing reflux extraction.

CN105693675 discloses a method for extraction of flavonoids from marigold containing ethanol dissolution or refluxing followed by one-dimensional chromatography and two-dimensional chromatography.

US7622599 discloses a process for isolating and purifying carotenoids containing higher concentrations of carotenoids. The process involves ensilaging Marigold flowers, dehydration, solvent extraction, alkali hydrolysis of carotenoid esters with absolute alcohol, crystallization/purification using water and absolute alcohol mixture followed by filtration and drying.

US8481769 discloses a process for isolation of carotenoids crystals having lutein and zeaxanthin in a weight ratio of about 10:1, 5:1 or 1:1. The process comprises obtaining an oleoresin rich in lutein and an oleoresin rich in zeaxanthin and mixing the two separately in a ratios ranging from about 80:20 (w/w) to 90:10 (w/w) or about 70:30 (w/w) to 30:70 (w/w) or about 10:90 (w/w) to 20:80 (w/w) and homogenized to obtain a mixed oleoresin.

EP3822257 discloses a process for extracting and isolating lutein crystal from vegetable oil resin. The process uses lipases for hydrolyzing the lutein fatty acid ester in vegetable oil resin to obtain lutein solution. The lutein is extracted using alcohol.

CN107475343 discloses a method for extracting lutein from marigold plants includes fermentation, enzymolysis, supercritical CO2 extraction and vacuum freeze-drying.

CN108277253 discloses a method for extracting zeaxanthin and lutein from corn and its processing by-products by using biological enzymes.

A research article titled, “Enzyme-mediated solvent extraction of carotenoids from marigold flower (Tagetes erecta)”, by E Barzana et. al. discloses a method of enzyme mediated solvent extraction of carotenoid esters from marigold petals. The method deals with the application of enzymes simultaneously with solvents to extract carotenoids esters.

An article titled, “Improving Xanthophyll Extraction from Marigold Flower Using Cellulolytic Enzymes”, by José Luis Navarrete-Bolaños et. al. discloses the use of cellulolytic enzymes from microbial consortium for hydrolysis of marigold petals.

An article titled, “Use of liquefied cold temperature dimethyl ether for extraction of pigments from fresh vegetable tissues” by A Noriyasu et. al. discloses the extraction of chlorophylls and carotenoids from green peel and yellow cortex from Japanese squash, spinach leaves and carrot roots using low temperature liquefied Dimethyl ether (DME). It further discloses the use of DME as a “green” solvent, as opposed to conventional solvents, for the preparation and extraction of various plant pigments.

An article titled, “Production of free lutein by simultaneous extraction and de-esterification of marigold flowers in liquefied dimethyl ether (DME)–KOH–EtOH mixture” by Panatpong Boonnoun et. al. discloses production of free lutein by simultaneous extraction and de- esterification of marigold flowers in liquefied Dimethyl ether-KOH-EtOH mixture using a chemical process which uses Dimethyl Ether for extraction of lutein esters and KOH-alcohol for the hydrolysis of esters. However, this process results in extremely low yield of free lutein and hence is not suitable for the production on large scale. Further, there is no mention of extraction of polyphenols and quercetagetin in this article. Even though this process discloses simultaneous extraction and de- esterification/ saponification in a single vessel to give free lutein, however it is not possible to separate flavanoids after saponification using this process.

An article titled, “Enzymatic hydrolysis of carotenoid esters of marigold flowers (Tagetes erecta L.) and red paprika (Capsicum annuum L.) by commercial lipases and Pleurotus sapidus extracellular lipase” by Holger Zorn et. al. (Volume 32, Issue 5, 8 April 2003, Pages 623-628) discloses a process for extraction of lutein from marigold and capsanthin from paprika and a process for enzymatic hydrolysis of the corresponding esters.

Another article titled, “Using Cellulases and Pectinases to Improve Better Extraction of Carotenoids from the Marigold Flowers (Tagetes erecta L.)” by Andrea et al. discloses use of enzymes for carotenoid extraction from Marigold Flowers (Tagetes erecta L.) and compares the yield of carotenoid extraction, using different enzyme concentration at different hydrolysis time. The process uses cellulases, hemicellulases and pectinases for hydrolysis of carotenoids mixture from the Marigold Flowers.

Most of the existing prior art processes for producing Marigold Oleoresin involves ensilation of fresh Marigold flowers having 88-92% moisture for 5 to 20 days (as per local practice) in anaerobic conditions. The ensilaged biomass is de-watered, shredded and dried in hot air at medium temperatures (60-90ºC) to reduce the moisture content to 10-12 % to render the same suitable for downstream extraction by organic solvents. The dried biomass, called Marigold Meal, is pelletised and then extracted by organic solvents like n-hexane, n-butane to produce Marigold Oleoresin. Alkali saponification of the Marigold oleoresin at 45-70ºC for 1 to 10 hours in an alcoholic medium result in cleaving of the ester bonds to release the Lutein and Zeaxanthin carotenoids. The carotenoid powders are isolated from the saponification mixture to give Lutein which is further purified with polar and/or non-polar solvents to ultimately reach 60- 80% purity level.

Xanthophylls are highly unstable compounds and are susceptible to oxidation on exposure to light, heat and air. Xanthophylls are lost during the various processing stages due to heat and oxidative process conditions. Apart from degradation and loss of Xanthophylls, Lutein, the most valuable pigment of the Marigold Xanthophylls, gets converted to Epoxides during the multi-step processing.

Some of the major drawbacks of the existing prior art processes for extracting bioactives from Marigold are-
1. The processes are multistage and in each stage there is significant loss of xanthophylls due to oxidation.
2. The total loss of Xanthophylls from fresh marigold to final product is around 30-40%.
3. The processes leave detectable range of organic solvent residues, mainly Hexane.
4. The processes require petrochemical solvents for efficient extraction.
5. Higher epoxide levels are present in the final products.
6. Complete extraction of Lutein or its esters are not possible.
7. Carotenoid yields obtained are extremely low to commercialize the process on large scale.

Therefore, there is a need to provide a commercial process for production and isolation of bioactives from Marigold flower which overcomes the aforementioned drawbacks of the prior art processes.

Hence, the present inventors, have come up with a one step process for isolation of total marigold extract comprising bioactives from silaged Marigold flower. Further isolation and purification of bioactives viz., Lutein and Quercetagetin achieved using green solvents, which is fast, easy, economical, ecofriendly and results in substantial increase in the yield of Lutein and Quercetagetin.

OBJECT OF INVENTION:
It is therefore an object of the present invention to provide a green process for isolation of total marigold extract from silaged Marigold flower using Dimethyl ether (DME) as the sole extraction solvent.

It is another object of the present invention to provide an extract having all the valuable components of Marigold flower which can be further fractionated into commercially valuable fractions like Oleoresin with high Lutein ester and flavonoids rich in quercetagetin in higher yields.

It is another object of the present invention to provide a process for isolation and purification of bioactives, Lutein and Quercetagetin from the total marigold extract.

It is yet another object of the present invention to provide Lutein and Zeaxanthin esters free of petrochemical solvents like hexane which can be used in the feed, food and nutraceutical industries and quercetagetin used in nutraceutical and personal care industry.

SUMMARY OF THE INVENTION:
In line with the above objectives, the present invention provides a process for isolation of total marigold extract comprising bioactives rich in lutein ester using DME as the sole extraction solvent.

Accordingly, the present invention provides a process for isolation of total marigold extract, wherein the said process comprising the steps of;
a. Mixing the fresh marigold flowers with an optimal mix of enzymes;
b. Simultaneously ensilaging and enzymatically hydrolyzing the fresh flower biomass of step (a) for a period of about 5 to 10 days;
c. Extracting the Marigold bioactives from the enzyme hydrolyzed biomass of step (b) using Dimethyl Ether (DME) to obtain Total Marigold Extract (TME) containing flavonoids, xanthophylls and water-soluble components; and
d. Optionally fractionating the total marigold extract of step (c) with ethyl alcohol to yield a lipophilic fraction with higher xanthophyll & lutein content and lipophobic flavonoid fraction rich in Quercetagetin.

The total extract is characterized to have 10-12% Xanthophylls, of which 86-88% is Lutein and 7-8% of Quercetagetin.

The composition of Marigold Oleoresin of the present invention is 17-21% Xanthophylls, of which 14.4-18% is Lutein and 0.7-1.4% is Zeaxanthin.

This Marigold oleoresin can be used for isolation of Lutein, free of hexane residues. The ethanol fraction is distilled to yield crude flavonoids with 40-50% Quercetagetin.

The process of the present invention results in higher xanthophyll recoveries from the input biomass, with an average xanthophyll recovery of 97.8%.

In another aspect, the optimal mix of enzymes is selected from the group comprising acellulases, hemicellulases, cellobiohydrases, pectinases, galactomannanases, proteases and lipases or combinations thereof.

In another aspect, the present invention provides a process for isolation of Lutein crystals from lipophilic fraction, wherein the said process comprising the steps of;
a. Mixing the Total Marigold Extract with propylene glycol and passing through centrifugal separator;
b. Saponifying the top lipophilic phase rich in lutein ester at 45-70ºC using alkali resulting in cleaving of the ester bonds to release the free Lutein and Zeaxanthin; and
c. Isolating the carotenoid crystals formed from the saponification mixture to give free Lutein.

In another aspect, the present invention provides a process for isolation of Quercetagetin from lipophobic flavonoid fraction which comprises decolourisation of the lipophobic flavonoid fraction with activated carbon at 50 -80ºC to isolate pure Quercetagetin.

DETAILED DESCRIPTION OF THE INVENTION:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

In line with the object of present invention provided a process for isolation of total marigold extract comprising bioactives rich in lutein ester from Marigold flower by a green process using DME as the sole extraction solvent.

Source of biological material used in the invention: Marigold seeds are imported from Suwanphingkarn Co. Ltd., 46/111, Soi Kubon 41 Rd. Samwatawantok, Khlongsamwa, Bangkok, Thailand, 10510 and further cultivated through backward integration specifically from farmers of Karnataka and Tamil Nadu, India.

The “Total Marigold Extract (TME)” as referred herein in the entire specification comprises flavonoids, xanthophylls and water-soluble components.

The “Marigold oleoresin” as referred herein in the entire specification encompasses Oleoresin with high Lutein ester.

The “composite marigold flavonoid” as referred herein in the entire specification encompasses marigold flavonoid rich in quercetagetin.

In an embodiment, the present process for isolation of the ensiled wet Marigold flowers using DME (Dimethyl Ether), yields an extract having all the valuable components of Marigold flowers in a single stage which can be further fractionated into commercially valuable fractions like Oleoresin with high Lutein ester and flavonoids rich in Quercetagetin.

Accordingly, the present invention relates to a process for isolation of total marigold extract from marigold flowers and further isolation and purification of bioactives, Lutein and Quercetagetin by subcritical extraction using dimethyl ether (DME), and ethanol, environment friendly green solvents.

In an embodiment, the present invention discloses a process for isolation of total marigold extract wherein the said process comprising the steps of;
a. Mixing the fresh marigold flowers with an optimal mix of enzymes;
b. Simultaneously ensilaging and enzymatically hydrolyzing the fresh flower biomass of step (a) for a period of about 5 to 10 days;
c. Extracting the Marigold bioactives from the enzyme hydrolyzed biomass of step (b) using Dimethyl Ether (DME) to obtain Total Marigold Extract (TME) containing flavonoids, xanthophylls and water soluble components; and
d. Optionally fractionating the total marigold extract of step (c) with ethyl alcohol to yield a lipophilic fraction with higher xanthophyll & lutein content and lipophobic flavonoid fraction rich in Quercetagetin.

In the above process, the silaged wet biomass goes through extraction process with DME. After removing DME the leftover biomass is the Total Marigold Extract. After fractionation with ethyl alcohol, the two products obtained are Lipophilic fraction containing 17-21% Xanthophyll concentration and Lipophobic flavonoid fraction containing 40-70% quercetagetin. Lipophilic fraction is further saponified with propylene glycol to produce free lutein with 80% concentration and the Lipophobic flavonoid fraction is further purified to produce quercetagetin.

The Marigold flowers considered for the extraction has Xanthophyll content of 0.16 -0.3% and moisture content of 88-92%.

In another embodiment, the optimal mix of enzymes as used in the present invention is selected from the group comprising cellulases, hemicellulases, cellobiohydrases, pectinases, galactomannanases, proteases, betaglucosidases, xylanases and lipases or combinations thereof. More preferably, the enzyme mix consists of combination of Betaglucosidases: Proteases: Xylanases in 2:1:1 ratio.

In another embodiment, the enzyme mix is used in an amount of 0.02-0.5% of fresh flower weight.

Dimethyl Ether (DME) has a low boiling point of -24ºC and so the solvent is flashed out from the miscella leaving behind a Total Marigold Extract (TME) with flavonoids, xanthophylls and water-soluble components.

Thus the total marigold extract obtained using DME has all the bioactives naturally present in the flowers with higher recoveries. The said extract has substantially higher concentration of Lutein in ester form along with flavonoids and polysaccharides.

In another embodiment, the present process provides simultaneous ensilation and hydrolysis of biomass i.e. Enzymatic Hydrolysis which is a highly efficient process that renders the biomass easy for direct extraction without the need to go through traditional ensilation, drying, grinding and palletization.

In another embodiment, the total marigold extract is further fractionated to polyphenols, water soluble components and xanthophyll fraction using ethyl alcohol or propylene glycol.

In another embodiment, the total marigold extract is fractionated to lipophilic fraction rich in xanthophylls and a lipophobic fraction having the flavonoids.

The fractionated total marigold extract yields a lipophilic fraction with higher xanthophyll & lutein content and flavonoid fraction rich in Quercetagetin.

The xanthophyll fraction can be de-esterified to yield high purity Lutein.

In another embodiment, the total marigold extract is characterized to have 10-12% Xanthophylls, of which 86-88% is Lutein and 7-8% of Quercetagetin.

In yet another embodiment, the composition of lipophilic fraction of the present invention comprises of 17-21% Xanthophylls, of which 14.4-18% is Lutein and 0.7-1.4% is Zeaxanthin.

The process of the present invention results in higher xanthophyll recovery amounting to an average of 97.8% of the input biomass xanthophyll.

In another embodiment, the Marigold oleoresin can be used for isolation of Lutein.

In another embodiment, the present invention discloses a process for isolation of Lutein crystals from lipophilic fraction, wherein the said process comprising the steps of;
a. Mixing the total Marigold Extract with propylene glycol and passing through centrifugal separator;
b. Saponifying the top lipophillic phase rich in lutein ester at 45-70ºC using alkali resulting in cleaving of the ester bonds to release the free Lutein and Zeaxanthin; and
c. Isolating the carotenoid crystals formed from the saponification mixture to give free Lutein.

In another embodiment, the alkali used for saponification is selected from the group comprising of Potassium hydroxide, Sodium hydroxide, Sodium carbonate, Ammonium hydroxide or their mixtures or in its anhydrous form.

The lipophilic fraction is saponified in propylene glycol to produce free lutein of 80% concentration.

The flavonoid fraction settled at the bottom phase can be used for Quercetagetin isolation. Water solubles retained in the middle phase along with propylene glycol are isolated.

In another embodiment, the present invention discloses a process for isolation of Quercetagetin from lipophobic flavonoid fraction which comprises decolourisation of the crude extract with activated carbon at 50 -80ºC to isolate pure Quercetagetin.

In another embodiment, the ethanol fraction is distilled to yield crude flavonoids with 40-70% Quercetagetin.

In another embodiment, the present invention discloses a process for isolation of total marigold extract comprising bioactives from marigold using DME as the sole extraction solvent thereby the total marigold extract obtained by the process of the present invention is free of residual solvents.

In the light of the complete enzymatic hydrolysis of the marigold flowers followed by extraction with DME solvent, the process results in higher yield of the Lutein and Quercetagetin. The use of optimal mix of enzymes facilitates hydrolysis of cell wall components of the flowers thereby facilitates the extractability of the bio-actives from the ensiled flowers.

In another embodiment, the present invention discloses a process for isolation of total marigold extract comprising bioactives from marigold wherein the extraction proceeds at lower temperatures and pressure.

In another embodiment, the present invention discloses a flavonoid/polyphenol mixture rich in quercetagetin isolated from marigold using only green solvents selected from DME, ethanol and water.

In yet another embodiment, the present invention discloses a process for production of highly enriched quercetagetin (>90% purity) isolated from marigold flower.

In another embodiment, the present invention discloses production of hexane free lutein from marigold flower using green solvents selected from DME and Ethanol.

In another embodiment, the present invention discloses a solvent free lutein produced from marigold flower using only DME green solvent and propylene glycol as processing aid.

In another embodiment, the present invention discloses a hexane free marigold oleoresin with high lutein concentration produced from marigold using only DME as a green solvent and free of any other organic solvents.

In another embodiment, the present invention discloses process to obtain marigold xanthophylls with higher lutein esters and separate quercetagetin rich marigold polyphenols. By the process of the present invention, it is possible to obtain quercetagetin in a purity of 90% or higher.

In another embodiment, the yield of lutein obtained by the present process is 10% of the Total Marigold Extract (TME) and 20% of the lipophilic fraction of TME.

In another embodiment, the yield of quercetagetin obtained by the present process is 60% of the lipophobic fraction of TME

In another embodiment, the present invention discloses a unique wet extraction process using DME which eliminates the energy intensive drying steps, maximizes xanthophyll recovery, minimizes epoxide formation and ultimately produces Marigold oleoresin of higher lutein content.

In another embodiment, the present invention discloses a process for isolation of total marigold extract comprising bioactives from marigold wherein the process does not involve the use of any petrochemical solvent at any stage of the process and downstream processing.

In another embodiment, the Lutein and Zeaxanthin esters thus produced by the present process are free of petrochemical solvents like hexane.
In an embodiment, the Lutein and Zeaxanthin esters obtained by the process of the present invention can be used in the feed, food and nutraceutical industries and quercetagetin obtained by the process of the present invention can be used in nutraceutical and personal care industry.

In another embodiment, the isolated Lutein crystals provided according to the present invention can be used as nutritional supplements, eye care supplements and pharmaceutical compositions.

The present process has the following advantages/benefits–
1. The total marigold extract obtained by the process of the present invention contains all the bioactives naturally present in the flowers with higher recoveries.
2. The process is a unique extraction process which bypasses the traditional industrial method of drying and pelletizing resulting in energy savings and lesser oxidation loss.
3. The process gives Marigold oleoresin with higher Xanthophyll content and lesser epoxides.
4. The process results in production of Lutein and Zeaxanthin esters and quercetagetin which are completely free of hexane and any other solvents.
5. The process is a green process for isolation of Lutein ester from the silaged marigold flower without using harmful organic solvents like hexane.
6. The entire process of isolation of lutein and quercetagetin from fresh marigold flowers is environment friendly using only green solvents such as Dimethyl Ether, Propylene glycol and ethanol.
7. The process results in high yield production of lutein ester and quercetagetin that can be scaled up to commercial levels.

The following example, which includes preferred embodiments, will serve to illustrate the
practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Examples:
Example 1:
1 kg of enzyme hydrolysed biomass having 1.2 % xanthophylls and 60% moisture was charged to the 20 L extraction vessel. An internal basket was used for holding solid material, the feed material was loaded into the basket. The basket was inserted into the extraction vessel and sealed with a spring loaded seal against the vessel wall at the top edge of the basket. Liquid dimethyl ether (DME) was circulated from a room temperature to supply bottle. DME was sub- cooled to 2 deg C before being pumped with an air driven piston pump up to operating pressure. DME was then pre heated to the extraction pressure before passing in to the extraction vessel. During circulation of DME, pressure was maintained in the extraction vessel using a flow control valve on the outlet of the vessel. After the flow control valve the DME pressure was reduced to the supply bottle pressure and heated to vaporize the DME. Vaporised DME was passed into the separation vessel to allow dissolved material to separate. Extraction was carried out using 1 kg biomass at varying pressure and temperature to validate the process.

Table 1:
Trials Feed Material Qty (Kg) Solvent Volume Extraction Temperature Extraction Pressure (bars) Extraction Time ( hr) Product ( Total Marigold Extract from ensilaged biomass )

Yield (%)

Xanthophyll %

Lutein %

Quercetagetin % Extraction Recovery % of xanthophylls
1 1 15 45-50 deg C 6-7 8 9.9 10.1 86 7.6 83.3
2 1 15 40- 45 deg C 9-10 8 10 11 86 7.4 91.6
3 1 15 36- 38 deg C 9-10 8 10.8 10.9 88 8.2 98.1

Example 2 :-
Following the procedure described above, 9kg of enzyme hydrolysed biomass was charged to the extraction vessel and dimethyl ether was used as the extraction solvent. Dimethyl ether was used in its liquefied state at 10 bars of pressure and 37 deg Celsius. A quantity of 135 kg of dimethyl ether was passed through the extraction vessel, collected extract was centrifuged to separate the water layer and then Total Marigold Extract was collected. The spent biomass was then recovered from the vessel and the extract and the spent biomass were analysed for the xanthophyll, Lutein and flavonoid Quercetagetin content. Extraction was carried out using 9 kg biomass at same parameters to validate the recovery.

Table 2:
Trials Sample Weight in kgs Xanthophyll % Lutein % Quercetagetin % Extraction Recovery %
Trial 1 Input enzyme hydrolysed biomass 9 1.2 87 1.1

97.2
Extract 1 10.5 87 8.1
Spent biomass 7.5 0.03 Not detected Not detected
Trial 2 Input enzyme hydrolysed biomass 9 1.2 87 1.1

98.2
Extract 1.04 10.2 87.5 8.5
Spent biomass 7.4 0.02 Not detected Not detected
Trial 3 Input enzyme hydrolysed biomass 9 1.2 87 1.1

98.1
Extract 1 10.6 87 8.7
Spent biomass 7.6 0.02 Not detected Not detected

Substantially, all of the xanthophyll from the charge of input biomass was recovered in the extract (Table 2), the average extraction recovery was 97.8%.

Example 3 :-
1 kg of the Total Marigold Extract fractionated with 10 volumes of Ethyl alcohol yielded lipophilic & lipophobic fractions. The lipophilic fraction had the superior marigold oleoresin with 20% xanthophylls & 87% Lutein. Lipophobic fraction after purification with 5 volumes of water yielded marigold flavonoid extract with 48 to 50% quercetagetin (Table 3). The composition of marigold flavonoid extract thus obtained is shown in table 6.

Table 3:
Total Marigold Extract ( kg ) Lipophilic fraction Lipophobic fraction
Yield % Xan % Lutein% Yield % QG%
1 60% 20% 87 11 48

Example 4 :-
1 kg of the marigold flavonoid extract was decolourized with 1 kg activated carbon at 70 deg C in a vessel under stirring for 1 hr. Carbon treated miscella was filtered on 0.1 Micron filter paper, after desolventization yielded 90% pure Quercetagetin with 60% yield.

Example 5 :-
1 kg of Total Marigold extract was mixed with 3 volumes of propylene glycol and passed through the centrifugal separator. Upper phase marigold oleoresin was separated. Alkali saponification of the xanthophyll extract at 70 deg C for 10 hr in propylene glycol resulted in cleaving of the ester bond to release free lutein in more than and 80% concentration and with more than 10% yield. Table 4 shows the analytical results.

Table 4
Trial Total Marigold Extract ( Kg) Solvent free Lutein crystals
Yield % Lutein Concentration %
1 1 10 82
2 1 10.6 80.6
3 1 10.2 81

Example 6 :- Alkali saponification of the Lipophilic fraction of the Total Marigold extract at 70 deg C for 10 hr in propylene glycol resulted in cleaving of the ester bond to release free lutein in more than 80% concentration and in about 20% yield. Table 5 shows the analytical results.

Table 5
Trial Weight of the fraction ( Kg) Hexane free Lutein crystals
Yield % Lutein Concentration %
1 1 20.45 81.4
2 1 21.13 82
3 1 19.96 82.4

Example 7 :- Composition of Marigold flavonoids obtained by the process of examples 1 and 2
Table 6
Composition of marigold Flavanoids
Quercetagetin % 50
Beta- sitosterol % 0.5
Stigmasterol% 0.25
Galangin % 0.003
Kaempferol % 0.005
6-Hydroxykaempherol % 3
Quercetin % 0.5
Patuletin % 5.5
Beta- sitosterol - Beta -D-glycoside 0.25
Carbohydrate % 22
Protein % 5.1
Sugar % 1.23
Fat % 4.85
Ash % 2.5
Crude Fibre % 0.1
Moisture % 4.1
, Claims:
1. A process for isolation of total marigold extract using green solvent Dimethyl Ether (DME) as a sole solvent, comprising the steps of;
a. Mixing the fresh marigold flowers with an optimal mix of enzymes;
b. Simultaneously ensilaging and enzymatically hydrolyzing the fresh flower biomass of step (a) for a period of about 5 to 10 days;
c. Extracting the Marigold bioactives from the enzyme hydrolyzed biomass of step (b) using Dimethyl Ether (DME) to obtain Total Marigold Extract (TME) containing flavonoids, xanthophylls and water soluble components; and
d. Optionally fractionating the total marigold extract of step (c) with ethyl alcohol to yield a lipophilic fraction with higher xanthophyll & lutein content and lipophobic flavonoid fraction rich in Quercetagetin.
2. The process as claimed in Claim 1, wherein the said optimal mix of enzymes are selected from the group comprising acellulases, hemicellulases, cellobiohydrases, pectinases, galactomannanases, proteases and lipases or combinations thereof.
3. The process as claimed in Claim 1, wherein the enzyme mix is used in an amount of 0.02-0.5% of fresh flower weight basis.
4. The process as claimed in Claim 1, wherein the said total marigold extract is fractionated into xanthophyll fraction and flavonoid fraction using ethyl alcohol or propylene glycol.
5. The process as claimed in Claim 1, wherein the said xanthophyll fraction is de-esterified to yield high purity Lutein.
6. The process as claimed in Claim 1, wherein the said Total Marigold extract is characterized to have 10-12% Xanthophylls, of which 86-88% is Lutein and 7-8% of Quercetagetin.
7. The process as claimed in Claim 1, wherein the said the composition of lipophilic fraction comprises 17-21% Xanthophylls, of which 14.4-18% is Lutein and 0.7-1.4% is Zeaxanthin.

8. The process as claimed in Claim 1, wherein the said ethanol fraction is distilled to yield crude flavonoids with 40-70% Quercetagetin.
9. The process as claimed in Claim 1, wherein the Total Marigold extract obtained is characterized by Quercetagetin rich flavonoid fraction.
10. The process as claimed in Claim 1, wherein the said flavonoid fraction rich in quercetagetin is isolated from marigold using only green solvents selected from DME, ethanol and water.
11. The process as claimed in any one of the preceding claims, wherein the said extraction proceeds at lower temperatures and pressure.
12. The process as claimed in Claim 1, wherein the quercetagetin isolated from marigold flower has a purity of more than 90%.
13. The process for isolation of Lutein crystals from lipophilic fraction as claimed in claim 1, wherein the said process comprising the steps of;
a. Mixing the total Marigold Extract with propylene glycol and passing through centrifugal separator;
b. Saponifying the top lipophillic phase rich in lutein ester at 45-70ºC using alkali resulting in cleaving of the ester bonds to release the free Lutein and Zeaxanthin; and
c. Isolating the carotenoid crystals formed from the saponification mixture to give free Lutein.
14. The process as claimed in Claim 13, wherein the said lutein is isolated from marigold flower using green solvents, DME and propylene glycol as processing aid.
15. The process as claimed in Claim 13, wherein the said alkali used for saponification is selected from the group comprising of Potassium hydroxide, Sodium hydroxide, Sodium carbonate, Ammonium hydroxide or their mixtures or in its anhydrous form.
16. The process as claimed in Claim 13, wherein the said lutein is free of any organic solvent like hexane.
17. The process for isolation of Quercetagetin from lipophobic flavonoid fraction as claimed in claim 1; wherein said process comprises decolourisation of the crude extract with activated carbon at 50 -80ºC to isolate pure Quercetagetin.