WO2022153228A1 - Purification of phytochemical(s) from areca wash liquid - Google Patents

Purification of phytochemical(s) from areca wash liquid Download PDF

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Publication number
WO2022153228A1
WO2022153228A1 PCT/IB2022/050287 IB2022050287W WO2022153228A1 WO 2022153228 A1 WO2022153228 A1 WO 2022153228A1 IB 2022050287 W IB2022050287 W IB 2022050287W WO 2022153228 A1 WO2022153228 A1 WO 2022153228A1
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Prior art keywords
areca
column
liquid
wash
hic
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PCT/IB2022/050287
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French (fr)
Inventor
Ganesh VENKATRAMAN BHAT
Nagendra Prakash
Harish Kumar
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Venkatraman Bhat Ganesh
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Publication of WO2022153228A1 publication Critical patent/WO2022153228A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/889—Arecaceae, Palmae or Palmaceae (Palm family), e.g. date or coconut palm or palmetto
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/06—Specific process operations in the permeate stream
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/26—Further operations combined with membrane separation processes
    • B01D2311/2623—Ion-Exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/26—Further operations combined with membrane separation processes
    • B01D2311/2697—Chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration

Definitions

  • the instant disclosure is in the field of biochemical sciences, particularly towards bioseparation/purification of phytochemicals.
  • the disclosure relates to method for purification of phytochemicals from areca wash liquid (AWL) . More particularly, the present method provides purification of phytochemicals selected from alkaloids, polyphenols, triterpenes or any combination thereof, from AWL.
  • AWL wash liquid
  • Areca plant (Areca catechu Linn.) is a tropical plant primarily grown in parts of Southeast Asia. Betel nut or arecanut has been used as a masticator by humans since the 4 th century AD. Arecanut is chewed by nearly 15% of the world population or nearly 1.1 billion people. In different parts of the world, arecanut is consumed in different ways. In India, the arecanut is consumed along with the betel leaves (the leaves of plant related to pepper) along with CaCOs or lime forming a dark red mixture upon mastication. India is a major hotspot for the production of arecanut which accounts to 6.5 million arecanut farmers and dominates worldwide production by producing 57-60% of the total produce (7,45,860 tonnes in 2016). In India, Karnataka produces about 55% of the areca produce (4,16,290 tonnes in 2016) which accounts to nearly 5% of the state’s GDP.
  • Areca plant (Areca catechu) is largely focused for its importance/utility of arecanut derived from areca fruit. Given the importance of areca plant as an important agricultural plantation/resource, there is a need for exploring beneficial molecules/components which can be further utilized economically in pharmaceutical, cosmetics, food and nutraceutical industries. The present disclosure addresses said need.
  • FIGS 1 to 7 illustrate alternate methods of purifying phytochemicals (alkaloids and/or polyphenols) from areca wash liquid (AWL) according to the present disclosure.
  • FIGS 8 to 14 illustrate chromatograms obtained through analytical HPLC method for AWL, permeate obtained from membrane fdtration, pure fractions obtained from HIC and IEX based on methods described in Examples 2 to 8 and Figures 1 to 7, respectively as follows:
  • FIG 8 Chromatogram of AWL (raw material) -
  • Figure 8A Chromatogram of AWL detected at 280nm showing the peaks of the presence of polyphenols [Retention Time (RT) 9.775, 11.697, 12.937, 14.556, 16.847, 20.188 are the peaks for epigallocatechin (EGC), catechin, syringic acid, epicatechin, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) respectively];
  • Figure 8B Chromatogram of AWL detected at 216nm showing the peaks of the presence of alkaloids [Retention Time (RT) 2.57 is the arecoline peak].
  • Figure 9 Chromatogram of method results based on Example 2 and Figure 1 -
  • Figure 9A Chromatogram of pure fraction (IEX) of Arecoline detected at 216nm (RT 2.719);
  • Figure 9B Chromatogram of ultrafiltration (membrane filtration) permeate detected at 280nm indicating the presence of polyphenols (RT 9.702, 11.579, 12.859, 14.038, 16.711, 20.086 are the peaks for epigallocatechin (EGC), catechin, syringic acid, epicatechin, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) respectively);
  • Figure 9C Chromatogram of pure fraction (HIC) of catechin detected at 280nm (RT 12.993);
  • Figure 9D Chromatogram of pure fraction (HIC) of EGC detected at 280nm (RT 9.639).
  • Figure 9E Chromatogram of pure fraction (HIC) of EGCG detected at 280nm (RT 16.573).
  • FIG. 10 Chromatogram of method results based on Example 3 and Figure 2 - Figure 10A: Chromatogram of ultrafiltration (membrane filtration) permeate detected at 280nm indicating the presence of polyphenols (RT 9.702, 11.579, 12.859, 14.438, 16.711, 20.086 are the peaks for epigallocatechin (EGC), catechin, syringic acid, epicatechin, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) respectively);
  • Figure 10B Chromatogram of ultrafiltration (membrane filtration) permeate detected at 216nm indicating the presence of alkaloids (RT 2.803 is the peak for arecoline);
  • Figure 10C Chromatogram of pure fraction (HIC) of EGC detected at 280nm (RT 9.911);
  • Figure 10D Chromatogram of pure fraction (HIC) of Catechin detected at 280nm (RT 12.926);
  • Figure 10E Chromatogram of pure fraction (HIC) of
  • FIG. 11 Chromatogram of method results based on Example 4 and Figure 4 - Figure 11 A: Chromatogram of ultrafiltration (membrane filtration) permeate detected at 280nm indicating the presence of polyphenols (RT 9.702, 11.579, 12.859, 14.438, 16.711, 20.086 are the peaks for epigallocatechin (EGC), catechin, syringic acid, epicatechin, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) respectively);
  • Figure 11B Chromatogram of ultrafiltration (membrane filtration) permeate detected at 216nm indicating the presence of alkaloid (RT 2.803);
  • Figure 11C Chromatogram of pure fraction (IEX) of Arecoline detected at 216nm (RT 2.708);
  • Figure 11D Chromatogram of pure fraction (HIC) of Catechin detected at 280nm (RT 12.926);
  • Figure HE Chromatogram of pure fraction (HIC) of Epicatechin detected at 280
  • FIG 12 Chromatogram of method results based on Examples 5 & 6 and Figures 4 & 5. The figure depicts chromatogram of pure fraction (IEX) of Arecoline detected at 216nm (RT 2.745).
  • Figure 13 Chromatogram of method results based on Example 7 and Figure 6 - Figure 13 A: Chromatogram of ultrafiltration (membrane filtration) detected at 280nm indicating the presence of polyphenols (RT 9.497, 11.017, 12.860 are the peaks for epigallocatechin (EGC), catechin and syringic acid respectively);
  • Figure 13B Chromatogram of ultrafiltration (membrane filtration) detected at 216nm indicating the presence of arecoline (RT 3.096).
  • Figure 14 Chromatogram of method results based on Example 8 and Figure 7 -
  • Figure 14A Chromatogram of pure fraction (HIC) of EGC detected at 280nm (RT 9.852)
  • Figure 14B Chromatogram of pure fraction (HIC) of Catechin, Epicatechin detected at 280nm (RT 12.912, 14.534)
  • Figure 14C Chromatogram of pure fraction (HIC) of EGCG detected at 280nm (RT 16.898).
  • arecanut As used in the present disclosure, the terms “arecanut”, “areca kernel”, “areca seed” or “betel nut” are used interchangeably and refers to the seed of the areca plant (Areca catechu) obtained by de-husking areca fruit.
  • whole arecanuts or “whole nuts” are used interchangeably and refers to the use of whole nuts of the areca fruit. In other words, said whole nuts are not cut/shredded into pieces and are used in their complete form/shape.
  • the phrase “at least one of alkaloid, polyphenol and triterpene” refers to the isolation/purification of alkaloid(s) alone; polyphenol(s) alone; triterpene(s) alone; alkaloid(s) and polyphenol(s); alkaloid(s) and triterpene (s); polyphenol(s) and triterpene(s); or all of alkaloid(s), polyphenol(s) and triterpene(s).
  • the present disclosure aims at addressing the need of identifying and isolating value-added components/phytochemicals from alternate agricultural plants/resources, particularly areca plant.
  • the present disclosure relates to purification of value-added components/molecules from areca wash liquid (AWL).
  • ADL wash liquid
  • the present disclosure particularly relates to a method for purification of phytochemicals from areca wash liquid (AWL), said method comprising subjecting the areca wash liquid (AWL) to membrane filtration, chromatography, or a combination of membrane filtration and chromatography; wherein the phytochemical is at least one of alkaloid, polyphenol and triterpene.
  • the method comprises purification of alkaloids selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof.
  • the method comprises purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
  • polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
  • the method comprises purification of triterpene which is ursolic acid, isomers of ursolic acid, or a combination thereof.
  • the areca wash liquid is a liquid composition obtained by processing areca fruit of the areca 15 plant.
  • the areca wash liquid refers to the liquid composition obtained by processing raw arecanuts, wherein the raw arecanuts are boiled in water at boiling temperature at atmospheric pressure for about 1 hour to 12 hours, preferably 2 hours to 5 20 hours.
  • the areca wash liquid refers to the liquid composition obtained by processing ‘whole arecanuts’ without cutting/shredding said whole arecanut into pieces.
  • the whole arecanut remains intact while processing the whole
  • AOL isca wash liquid
  • the areca wash liquid refers to the liquid composition obtained by a process comprising: a) dehusking areca fruits to obtain arecanuts; kB)O boiling the arecanuts (whole nuts) in presence of a solvent at a temperature of about 50°C to about 100°C for a time-period ranging from about 1 hour to 12 hours, preferably 1 hour to 5 hours, wherein the solvent is selected from a group comprising water, a polar solvent, or a combination thereof; c) separating the liquid remnant/liquid composition from the arecanuts after step b), wherein said liquid composition is the areca wash liquid.
  • the areca wash liquid refers to the liquid composition obtained by a 5 process comprising: a) de-husking areca fruits to obtain arecanuts; b) boiling the arecanuts (whole nuts) in presence of a solvent at a temperature of about 50°C to about 100°C for a time-period ranging from about 1 hour to 12 hours, preferably 1 hour to 5 hours; and d)0 separating the liquid remnant from the arecanuts after step b), d) boiling fresh de-husked arecanuts (whole nuts) in presence of the solvent, the separated liquid remnant of step c), at a temperature of about 50°C to about 100°C for a time-period ranging from about 1 hour to 12 hours, preferably 1 hour to 5 hours, e) separating liquid remnant from the arecanuts after step d), wherein said liquid remnant is the 15 areca wash liquid (AWL), wherein the solvent is selected from a group comprising water, a polar solvent, or a combination thereof
  • the solvent in above step b) or step d) is water.
  • the solvent in step b) or step d) is a polar solvent.
  • the solvent in above step b) or step d) is a polar solvent selected from a group comprising ethanol, methanol, ethyl acetate, isopropyl alcohol (IPA) and 25 combinations thereof.
  • the process of obtaining areca wash liquid comprises adding bases such as lime (calcium hydroxide) in addition to the solvent.
  • the boiling of whole arecanuts [above step b) - boiling the whole arecanuts in presence of a solvent] optionally comprises adding a quantity of previously 30 obtained or previously prepared areca wash liquid.
  • the boiling is carried out at atmospheric pressure.
  • the areca wash liquid refers to the liquid composition obtained by a process comprising: a) harvesting areca fruits from areca plant/tree; b) dehusking areca fruits to obtain arecanuts; c)5 boiling the arecanuts (whole arecanuts) in water at a temperature of about 65 °C to about
  • the areca wash liquid refers to the liquid composition obtained by a process comprising: a) harvesting areca fruits from areca plant/tree; b) de-husking areca fruits to obtain arecanuts; c) boiling the arecanuts (whole nuts) in water at a temperature of about 65 °C to about 100°C 15 and at atmospheric pressure for a time-period ranging from about 2 hours to 5 hours; and a) separating liquid from the arecanuts after step c), to obtain separated liquid and boiled arecanuts, d) boiling fresh de-husked arecanuts (whole nuts) in water and the separated liquid of step d), at a temperature of about 65 °C to about 100°C and at atmospheric pressure for a time -period
  • step e) separating liquid from the arecanuts after step e), wherein said liquid is the areca wash liquid (AWL), and wherein said step e) and step f) are optionally repeated for about 1 to 5 times.
  • ADL areca wash liquid
  • the areca wash liquid refers to the liquid composition obtained by a process comprising: a) harvesting areca fruits from areca plant/tree; b) dehusking areca fruits to obtain arecanuts; c) boiling the arecanuts (whole nuts) in presence of a polar solvent selected from a group 30 comprising ethanol, methanol, ethyl acetate and combinations thereof, at a temperature of about 50°C to about 85°C and at atmospheric pressure for a time -period ranging from about 1 hour to 3 hours; and d) separating liquid and arecanuts, wherein said liquid is the areca wash liquid.
  • a polar solvent selected from a group 30 comprising ethanol, methanol, ethyl acetate and combinations thereof
  • the areca wash liquid refers to the liquid composition obtained by a process comprising: a) harvesting areca fruits from areca plant/tree; b) de-husking areca fruits to obtain arecanuts; c)5 boiling the arecanuts (whole nuts) in presence of a polar solvent at a temperature of about
  • step e) separating liquid from the arecanuts after step c), to obtain separated liquid and boiled arecanuts, d)0 boiling fresh de-husked arecanuts (whole nuts) in a polar solvent and the separated liquid of step d), at a temperature of about 50°C to about 85°C and at atmospheric pressure for a timeperiod ranging from about 1 hour to 3 hours, at a temperature of about 65 °C to about 100°C and at atmospheric pressure for a time-period ranging from about 2 hours to 5 hours; and f) separating liquid from the arecanuts after step e), wherein said liquid is the areca wash liquid 15 (AWL), wherein the polar solvent is selected from a group comprising ethanol, methanol, ethyl acetate and combinations thereof, and wherein said step e) and step f) are optionally repeated for about 1 to 5
  • the areca wash liquid refers to the liquid composition obtained by a method comprising steps of: a) harvesting areca fruits from areca plant/tree; b) dehusking areca fruits by pealing/removing the outer fibrous husk of the areca fruits to obtain arecanuts;
  • ⁇ 2)5 boiling the arecanuts (whole nuts) in a vessel or a tank in the presence of a solvent selected from a group comprising water, ethanol, methanol, ethyl acetate and combinations thereof, along with mild addition of a base such as calcium hydroxide at concentration ranging from about 0.5 to 7.5% w/v, at a temperature of about 55°C to about 100°C and at atmospheric pressure for a time-period ranging from about 1 hour to 4 hours, and wherein said boiling 30 optionally comprises adding a quantity of previously obtained areca wash liquid; and d) separating liquid and arecanuts, wherein said liquid is the areca wash liquid.
  • a solvent selected from a group comprising water, ethanol, methanol, ethyl acetate and combinations thereof, along with mild addition of a base such as calcium hydroxide at concentration ranging from about 0.5 to 7.5% w/v, at a temperature of about 55°C to about 100°C and at atmospheric pressure for a time
  • the areca wash liquid refers to the liquid composition obtained by a process comprising: a) harvesting areca fruits from areca plant/tree; b) de-husking areca fruits by pealing/removing the outer fibrous husk of the areca fruits to obtain arecanuts; c) boiling the arecanuts (whole nuts) in a vessel or a tank in presence of a solvent along with 5 mild addition of a base at concentration ranging from about 0.5 to 7.5% w/v, at a temperature of about 55°C to about 100°C and at atmospheric pressure for a time-period ranging from about 1 hour to 4 hours, d) separating liquid from the arecanuts after step c), to obtain separated liquid and boiled arecanuts, d)0 boiling fresh de-husked arecanuts (whole nuts) in presence of a solvent and the separated liquid of step d) along with mild addition of a base at concentration ranging from about 0.5 to 7.5% w/v, at a temperature
  • the above described process of obtaining the areca wash liquid further comprises optionally agitating the vessel or tank comprising arecanuts and solvent during the boiling of the arecanuts, wherein said agitation is for one or more times.
  • the areca wash liquid refers to the liquid composition essentially comprising: a) polyphenols, b) alkaloids, c) polysaccharide, d) saponins, e) triterpenes, f) steroids, g) minerals, and h) optionally, a residual component selected from a group comprising sugars, proteins, tannins aggregates, fat, impurities and combinations thereof.
  • the areca wash liquid refers to the liquid composition essentially comprising: a) polyphenols, b) alkaloids, c) saponins, d) triterpenes, e) steroids, f) polysaccharide, g) minerals, and h) a residual component selected from a group comprising sugars, proteins, tannin aggregates, fat, impurities and combinations thereof.
  • the areca wash liquid essentially comprises: a) polyphenols selected from catechin, epigallocatechin gallate (EGCG), epicatechin isomer of EGCG, isomer of catechin, isomer of epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), syringic acid, resveretrol, quercetin, gallic acid, coumaric acid, leucocyanidins, dimeric and complex cyanidins, luteolin, liquiritigenin, chrysoeriol, isorhamnetin, or any combination thereof; b) alkaloids selected from arecoline, guvacine, arecaidine, guvacoline or any combination thereof; c) triterpenes and steroids selected from ursolic acid, sitosterol, arborinol, cylindrin, femenol, cycloartenol, ergosterol, isomers thereof, or any combination thereof;
  • the areca wash liquid when dehydrated and analyzed essentially comprises polyphenols at a concentration ranging from about 15 wt % to about 70 wt %, alkaloids at a concentration ranging from about 0.5 wt % to about 4.5 wt %, polysaccharide at a concentration ranging from about 12 wt % to about 30 wt %, minerals at a concentration ranging from about 0.2 wt % to about 2 wt %, and residual component at a concentration ranging from about 0.3 wt % to about 1.5 wt %.
  • the ingredients/components of the areca wash liquid (AWL) add up to 100 wt%.
  • the areca wash liquid comprises the following characteristics and composition:
  • the areca wash liquid also comprises additional components/molecules (apart from the areca wash liquid composition described above) and said additional components/molecules is based on the composition of arecanut.
  • the arecanut 5 composition comprises polyphenol (45-70 wt %), fat (15-19 wt %), starch (20-25 wt %) and alkaloids (1.5-4.5 wt %). Further characterization of the arecanut composition is as follows:
  • polyphenols present in arecanut are mostly flavonols, that include about 12-30% of catechin, about 8-20% epicatechin, about 12-17% of leucocyanidin, the remaining portion being complex flavonoids in varying degrees of polymerization.
  • dimeric, trimeric and tetrameric procyanidins have been isolated from arecanut. These are mainly, epigallocatechin gallate (about 3-15%), catechin gallate (about 2-10%), epicatechin gallate. Quercetin, coumaric acid and gallic acid are also present.
  • Alkaloids The four major alkaloids present in arecanut are arecoline (about 7.5 to 21.5 mg/g), arecaidine (about 1.5 to 6.2 mg/g), guvacoline (about 2.0 to 5.0 mg/g) and guvacine
  • Arecoline is a colorless volatile alkaloid.
  • Triterpenes and steroids 1-6% dry weight of arecanut comprises of these class of molecules such as ursolic acid, femenol, sitosterol, ergosterol, cylindrin and other traces.
  • Fats consist of about 15 to 19% dry weight of arecanut.
  • Arecanut fatty acid profile 20 comprises about 19.5% lauric acid, about 46.2% myristic acid, about 12.7% palmitic acid, about 1.6% stearic acid, about 0.3% decanoic acid, about 6.2% oleic acid, about 5.4% dodecenoic acid, about 0.3% tetra decanoic acid and about hexadecenoic acid 7.2%.
  • Starch is the primary polysaccharide and accounts for about 20 to 25% of polysaccharides.
  • the mineral content primarily includes calcium, phosphorus and iron. It also contains traces of Vitamin B6 and Vitamin C.
  • concentrations of the components in the areca wash liquid vary based on the types of solvent (water or an alcoholic solvent) employed for obtaining areca wash liquid. For instance, employing solvents such as ethanol or methanol may affect the yield of the components in the areca wash liquid, wherein the yield may increase by 1.5 to 2 times when compared to the areca wash liquid obtained by employing water as a solvent.
  • AWL red coloured areca wash liquid
  • Chogaru in Kannada
  • Kalipak in Malayalam
  • the areca wash liquid employed in the present disclosure is the liquid composition obtained by the process described above and/or the liquid composition comprising the composition as described above. Any variations in the process of obtaining areca wash liquid and/or the composition areca wash liquid as described herein is well within the purview of a skilled person in the art. Particularly, the areca wash liquid may also be obtained by any other areca fruit processing method(s) as long as the obtained areca wash liquid comprises phenols and/or alkaloids.
  • the areca wash liquid may also be obtained by any other areca fruit processing method(s) as long as the obtained areca wash liquid comprises one or more of arecoline, arecaidine, guvacine, guvacoline, epigallocatechin gallate (EGCG), catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), syringic acid, coumaric acid, resveretrol, ursolic acid, and/or isomers of said compounds.
  • EGCG epigallocatechin gallate
  • catechin epicatechin
  • epigallocatechin ECG
  • ECG epicatechin gallate
  • ECG epicatechin gallate
  • syringic acid coumaric acid
  • resveretrol resveretrol
  • ursolic acid and/or isomers of said compounds.
  • the present disclosure thus relates to a method for purification of phytochemicals from areca wash liquid (AWL).
  • ADL areca wash liquid
  • the present disclosure describes a method for purification of phytochemicals selected from a group comprising polyphenols, alkaloids, triterpenes, and combinations thereof, from areca wash liquid (AWL).
  • the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline and combinations thereof, from areca wash liquid (AWL).
  • the present disclosure describes a method for purification of polyphenols selected from catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL).
  • the present disclosure describes a method for purification of triterpenes selected from a group comprising ursolic acid, its isomers, and a combination thereof, from areca wash liquid (AWL).
  • ADL areca wash liquid
  • the present disclosure relates to a method for purification of alkaloids and polyphenols as described above.
  • the present disclosure describes a method for purification of phytochemicals selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from AWL.
  • the present disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), said method comprising subjecting the areca wash liquid to membrane filtration, chromatography, or a combination of membrane filtration and chromatography.
  • the method additionally comprises employing polar and non-polar solvents aiding the purification/separation processes.
  • the present disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), said method comprising subjecting the areca wash liquid to membrane filtration and chromatography in any order.
  • ADL areca wash liquid
  • the present disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), said method comprising subjecting the areca wash liquid to membrane filtration, chromatography, or a combination of membrane filtration and chromatography in any order, wherein the phytochemical is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
  • ABL areca wash liquid
  • the chromatography is ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), or a combination of both.
  • the ion exchange chromatography is cation exchange chromatography or anion exchange chromatography.
  • the cation exchange chromatography purifies at least one alkaloid selected from arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination of alkaloid compounds thereof.
  • anion exchange chromatography can be used as a standalone or additional chromatographic technique for the purification of same alkaloids.
  • the hydrophobic interaction chromatography purifies at least one polyphenol selected from the group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof; and/or triterpenes (ursolic acid or isomers thereof).
  • the hydrophobic interaction chromatography employed here could be understood as a chromatography which works by adsorption of molecules such as the above described polyphenols and other such molecules, and desorption and elution based on polarity, hydrophobicity and optionally accompanied by a charge/ionic interaction.
  • the membrane filtration is selected from cross flow filtration (CFF), depth filtration, or a combination thereof.
  • the membrane filtration employed is ultrafiltration and/or microfiltration, based on the principles/mechanism of cross flow filtration (CFF).
  • the membrane filtration separates large molecules selected from at least one of sugars, proteins, tannin aggregates, fats, other high molecular weight components, impurities, and combinations thereof.
  • the areca wash liquid is optionally subjected to pre-filtration using Whatman filter paper, depth filter, centrifugation, microfiltration, ultrafiltration, continuous centrifugation or any combination thereof, before subjecting the areca wash liquid to membrane filtration, chromatography, or the combination of membrane filtration and chromatography .
  • the present disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), said method comprising subjecting the AWL to at least one of membrane filtration, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography (HIC), or any 15 combination of membrane filtration, cation exchange chromatography, anion exchange chromatography and hydrophobic interaction chromatography (HIC) in any order, wherein the phytochemical is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers 20 of said compounds, and combinations thereof.
  • AWL areca wash liquid
  • HIC hydrophobic interaction chromatography
  • the present disclosure describes a method for purification of alkaloids and polyphenols from areca wash liquid (AWL), wherein the alkaloid is selected from the group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, 25 and combinations thereof, and the polyphenol is selected from the group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i. subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify alkaloid; 30 and ii. performing membrane filtration, followed by hydrophobic interaction chromatography
  • IEX ion exchange chromatography
  • the present disclosure describes a method for purification of alkaloids and polyphenols from areca wash liquid (AWL), wherein the alkaloid is selected from the group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, and the polyphenol is selected from the group comprising 5 catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i.
  • the method for purification of phytochemicals selected from a group 15 comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds and combinations thereof, from areca wash liquid (AWL) is illustrated in Figure 1.
  • the process determining/sensitive step according to the purification method of Figure 1 is the ion 20 exchange chromatography (IEX).
  • the present disclosure describes a method for purification of alkaloids and polyphenols comprising steps of: i) subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the 25 alkaloids, wherein the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, tB)O loading areca wash liquid (sample) onto the column and binding at a pH between 3.5 to 10.5, c) intermediate wash of the column with the equilibration buffer of step a) to obtain initial wash flow through liquid (IWFT) comprising unbound molecules, and d) elution of the column using an elution buffer of pH between 2 to 11 to replace the alkaloids with ionic groups, and obtaining an e
  • membrane filtration comprises steps of: a) equilibrating a polymeric membrane with solvent (mobile phase), b) applying the initial wash flow through liquid (IWFT) (sample) onto the membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, and c) washing and re-equilibrating the membrane,
  • IWFT initial wash flow through liquid
  • hydrophobic interaction chromatography comprises steps of: a) equilibrating a HIC column packed with macro porous polymeric resin using an equilibration buffer having a pH of 3.5 to 10.5, b) application of the permeate (sample) and intermediate wash using the equilibration buffer of step a), and d)5 elution using an elution buffer of pH of 2.5 to 10.5 to obtain an eluate comprising polyphenol; wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof; and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin 20 (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
  • EECG Epicatechin Gallate
  • EGCG
  • the present disclosure describes a method for purification of alkaloids and polyphenols from areca wash liquid (AWL), wherein the alkaloid is selected 25 from the group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, and the polyphenol is selected from the group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i30 subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the alkaloid, and obtaining initial wash flow through liquid (IWFT) and an eluate, wherein the ion exchange chromatography comprises steps of: a) equilibration with suitable buffer of pH between about 3.5-10.5, b) binding at a pH between about 3.5-1
  • the present disclosure describes a method for purification of alkaloids and polyphenols from areca wash liquid (AWL), wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate
  • ADL areca wash liquid
  • the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof
  • the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate
  • EGCG syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i. subjecting the areca wash liquid to membrane filtration followed by hydrophobic interaction chromatography (HIC) to purify the polyphenol; and ii. performing ion exchange chromatography (IEX) to purify the alkaloid.
  • HIC hydrophobic interaction chromatography
  • IEX ion exchange chromatography
  • the present disclosure describes a method for purification of alkaloids and polyphenols from areca wash liquid (AWL), wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, 5 and combinations thereof, and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i.
  • the present disclosure describes a method for purification of alkaloids and polyphenols comprising steps of: i) subjecting the areca wash liquid to membrane filtration followed by hydrophobic interaction chromatography (HIC) to purify the polyphenols, wherein the membrane filtration 20 comprises steps of: a) equilibrating a polymeric membrane with solvent (mobile phase), b) applying the areca wash liquid (sample) onto the membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, c) washing and re-equilibrating the membrane,
  • HIC hydrophobic interaction chromatography
  • hydrophobic interaction chromatography comprises steps of: a) equilibrating a HIC column packed with macro porous polymeric resin using an equilibration buffer having a pH of 3.5 to 10.5, b) application of the permeate (sample) onto the HIC column, and intermediate wash using the equilibration buffer of step a) to obtain initial wash flow through liquid (IWFT) comprising
  • IEX ion exchange chromatography
  • the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration 5 buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, b) loading the IWFT (sample) onto the column ,and binding at a pH between 3.5 to 10.5, c) intermediate wash of the column with the equilibration buffer of step a), and dl)0 elution using an elution buffer of pH between 2 to 11 to replace the alkaloids with ionic
  • the method for purification of phytochemicals selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, 20 epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL) is illustrated in Figure 2.
  • the process determining/sensitive step according to the purification method of Figure 2 is the hydrophobic interaction chromatography (HIC).
  • the present 25 disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), wherein the phytochemical is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i30 subjecting the areca wash liquid to membrane filtration followed by hydrophobic interaction chromatography (HIC) to purify catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, or combinations thereof, wherein the membrane filtration comprises steps of: a) equilibrating a membrane of about 20
  • the permeate being the component of interest and the retentate comprising of impurities
  • the hydrophobic interaction chromatography employs macro porous polymeric resins, and comprises steps of: aO)0 equilibration using mobile phase of suitable ionic strength, solvent strength of about 15% to 100% of polar solvent and a pH of 3.5 - 10.5, b) application of the permeate (sample), and intermediate wash using the equilibration mobile phase to obtain initial wash flow through liquid (IWFT) comprising unbound molecules, and c) elution at a suitable solvent (polar) strength of about 20%-100% at a suitable pH of about 15 2.5-10.5 to obtain an eluate comprising the polyphenol; and ii.
  • IWFT ion exchange chromatography
  • IEX ion exchange chromatography
  • the present disclosure describes a method for purification of alkaloid and polyphenol from areca wash liquid (AWL), wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate
  • ADL areca wash liquid
  • the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof
  • the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate
  • EGCG syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i. subjecting the areca wash liquid to membrane filtration followed by ion exchange chromatography (IEX) to purify the alkaloid; and ii. performing hydrophobic interaction chromatography (HIC) to purify the polyphenol.
  • IEX ion exchange chromatography
  • HIC hydrophobic interaction chromatography
  • the present disclosure describes a method for purification of alkaloid and polyphenol from areca wash liquid (AWL), wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, 5 and combinations thereof, and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i. subjecting the areca wash liquid to membrane filtration followed by ion exchange
  • IEX initial wash flow through liquid
  • eluate wherein the eluate comprises the alkaloid
  • HIC hydrophobic interaction chromatography
  • the present disclosure describes a method for purification of alkaloids and polyphenols comprising steps of: i) subjecting the areca wash liquid to membrane filtration followed by ion exchange chromatography (IEX) to purify the alkaloids, wherein the membrane filtration comprises steps of:
  • step a (00 intermediate wash of the column with the equilibration buffer of step a) to obtain initial wash flow through liquid (IWFT) comprising unbound molecules, d) elution of the column using an elution buffer of pH between 2 to 11 to replace the alkaloids with ionic groups, and obtaining an eluate comprising the alkaloid; and ii) subjecting the initial wash flow through liquid (IWFT) obtained from the intermediate wash of the ion exchange chromatography [step c)] to hydrophobic interaction chromatography (HIC) to purify the polyphenols, wherein the hydrophobic interaction chromatography (HIC) comprises steps of: a)5 equilibrating a HIC column packed with macro porous polymeric resin using an equilibration buffer having a pH of 3.5 to 10.5, b) application of the IWFT (sample) onto the HIC column, and intermediate wash using the equilibration buffer of step a), and c) elution of the H
  • the method for purification of phytochemicals selected from the group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), 20 syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL) is illustrated in Figure 3.
  • the present disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), wherein the phytochemical is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), epicatechin 25 gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i.
  • the membrane filtration comprises steps 30 of: a) equilibrating a membrane of about 20000kD-lkD or lesser m.w.c.o.
  • the ion exchange chromatography comprises steps of: a) equilibration with suitable buffer of pH between about 3.5-10.5, b) loading the permeate (sample) and binding at a suitable optimized pH between about 3.5- 10.5, c) intermediate wash with the same equilibration buffer to obtain initial wash flow through 10 liquid (IWFT) comprising unbound molecules, d) elution using a stronger (higher conductivity) buffer of pH between about 2-11 to replace the alkaloids with ionic groups, to obtain an eluate comprising the alkaloid; and ii. subjecting the IWFT obtained from the intermediate wash of the ion exchange
  • step c)] to hydrophobic interaction chromatography (HIC) using macro porous polymeric resins comprises to purify catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, or any combination thereof, wherein the hydrophobic interaction chromatography comprises steps of:
  • the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), comprising: i30 subjecting the areca wash liquid to membrane filtration; and ii. performing ion exchange chromatography (IEX) to purify the alkaloid.
  • the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), comprising: i. subjecting the areca wash liquid to membrane filtration and obtaining a permeate; and ii.5 subjecting the permeate to ion exchange chromatography (IEX), to obtain an eluate comprising alkaloid.
  • ADL i30 subjecting the areca wash liquid to membrane filtration
  • IEX ion exchange
  • the present disclosure describes a method for purification of alkaloid comprising steps of: i) 10 subjecting the areca wash liquid to membrane filtration and obtaining a permeate, wherein the membrane filtration comprises steps of: a) equilibrating a polymeric membrane with solvent (mobile phase), b) applying the areca wash liquid (sample) onto the membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, and d)5 washing and re-equilibrating the membrane, and ii) subjecting the permeate to ion exchange chromatography (IEX) to purify the alkaloids, wherein the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration
  • the method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL) is illustrated in Figure 4.
  • the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), comprising: i.
  • membrane fdtration comprises steps of: a)5 equilibrating a membrane of about 20000kD-lkD or lesser m.w.c.o. (polymeric make such as PES or PVDF either in the cassette or in the hollow fibre type) with suitable mobile phase of suitable solvent strength, conductivity and pH, b) applying areca wash liquid (sample) at an optimized permeate flow and TMP leading to separation, wherein the permeate being the component of interest and the retentate
  • IEX ion exchange chromatography
  • the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method 25 comprising subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the alkaloid.
  • ABL isoca wash liquid
  • IEX ion exchange chromatography
  • the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of 30 said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising subjecting the areca wash liquid to ion exchange chromatography (IEX) to obtain an eluate comprising the alkaloid.
  • ADL isoca wash liquid
  • IEX ion exchange chromatography
  • the present disclosure describes a method for purification of alkaloids comprising subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the alkaloids, wherein the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration 5 buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, b) loading the areca wash liquid (sample) onto the column and binding at a pH between 3.5 to 10.5,
  • the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof.
  • the method for purification of arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL) is illustrated in Figure 5.
  • the present disclosure describes a method for purification of alkaloid selected from arecoline, arecaidine, guvacine, 20 guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the alkaloid selected from arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, wherein the ion exchange chromatography comprises steps of:
  • the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising: i. subjecting the areca wash liquid to membrane fdtration; and ii. performing hydrophobic interaction chromatography (HIC) to purify the polyphenol.
  • GAC epigallocatechin
  • ECG Epicatechin Gallate
  • EGCG epigallocatechin gallate
  • syringic acid coumaric acid
  • resveretrol isomers of said compounds, and combinations thereof
  • the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid
  • ADL ADL 10
  • the method comprising: i. subjecting the areca wash liquid to membrane filtration and obtaining a permeate; and ii. subjecting the permeate to hydrophobic interaction chromatography (HIC) to obtain an eluate comprising the polyphenol.
  • HIC hydrophobic interaction chromatography
  • the present disclosure describes a method for purification of polyphenol from areca wash liquid (AWL), the method comprising steps of: i) subjecting the areca wash liquid to membrane filtration and obtaining a permeate, wherein the membrane filtration comprises steps of: a) equilibrating a polymeric membrane with solvent (mobile phase),
  • hydrophobic interaction chromatography comprises steps of: a) equilibrating a HIC column packed with macro porous polymeric resin using an equilibration buffer having a pH of 3.5 to 10.5, b) application of the permeate (sample) onto the column and intermediate wash of the column
  • step a 30 using the equilibration buffer of step a), and c) elution of the column using an elution buffer of pH of 2.5 to 10.5 to obtain an eluate comprising polyphenol; wherein the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
  • the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
  • the method for purification of polyphenol selected from a group
  • the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), 10 epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising: i. subjecting the areca wash liquid to membrane filtration and obtaining a permeate, wherein the membrane filtration comprises steps of: )5 equilibrating a membrane of about 20000kD-lkD or lesser m.w.c.o.
  • hydrophobic interaction chromatography HIC
  • macroporous hydrophobic polymeric resins to purify the polyphenol
  • the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising subjecting the areca wash liquid to hydrophobic interaction chromatography (HIC) to purify the polyphenol.
  • GAC epigallocatechin
  • ECG epicatechin gallate
  • EGCG epigallocatechin gallate
  • syringic acid coumaric acid
  • resveretrol isomers of said compounds, and combinations thereof
  • the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising subjecting the areca wash liquid to hydrophobic interaction
  • the present disclosure describes a method for purification of polyphenol from areca wash liquid (AWL) comprises subjecting the areca wash liquid to hydrophobic 15 interaction chromatography (HIC) to purify the polyphenols, wherein the HIC comprises steps of: a) equilibrating a HIC column packed with macro porous polymeric resin using an equilibration buffer having a pH of 3.5 to 10.5, b) application of the areca wash liquid (sample) on to the column, and intermediate wash of the 20 column using the equilibration buffer of step a), and c) elution of the column using an elution buffer of pH of 2.5 to 10.5 to obtain an eluate comprising the polyphenol; wherein the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), 25 syringic acid, coumaric acid, resveretrol, isomers
  • the method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), 30 epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL) is illustrated in Figure 7.
  • the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising subjecting the areca wash liquid to hydrophobic interaction chromatography (HIC) to purify the polyphenol, wherein the hydrophobic interaction chromatography employs macro porous polymeric resin(s), and comprises steps of: a)5 equilibration of the column using mobile phase of suitable ionic strength, solvent strength of about 15% to 100% of polar solvent and a pH of 3.5 - 10.5, b) application of areca wash liquid (sample) on to the column, and intermediate wash of the column using the equilibration mobile phase, and c) elution of the column at a suitable solvent (polar)
  • the membrane fdtration comprises microfdtration, ultrafdtration or a combination thereof. In some embodiments, the membrane fdtration comprises employing membrane fdters having pore size ranging from about 20000kD to IkD. In some 15 embodiments, the membrane fdtration is employed for separating molecules having a high molecular weight. In some embodiments, the membrane fdtration is employed for separating molecules having a molecular weight ranging from about 1 kDato about 20000 kDa. In some embodiments, the membrane fdtration separates sugars, proteins, fats, tannin aggregates, high molecular weight compounds, impurities, and combinations thereof. In some 20 embodiments, the membrane fdtration is cross flow fdtration (CFF) such as ultrafdtration
  • CFF cross flow fdtration
  • UF UF
  • MF microfdtration
  • the membrane fdtration is accompanied by depth fdtration to ease other separation and fdtration processes.
  • the crossflow fdtration is a membrane fdtration technique in which the starting solution/sample passes tangentially along the surface of the filter. A pressure difference across the filter drives components that are smaller than the pores through the filter. Components larger than the filter pores are retained and pass along the membrane surface.
  • the membrane fdtration technique employed in the purification of phytochemicals from areca wash liquid (AWL) in the present disclosure is microfdtration and ultrafdtration.
  • the membrane fdtration technique employed in the purification of phytochemicals from areca wash liquid (AWL) in the present disclosure is performed using the following parameters/conditions:
  • PES polyethersulfone
  • PS Hydrosart®
  • PVDF polyvinylidene fluoride
  • nylon membrane polytetrafluoroethylene
  • TMP Transmembrane pressure
  • the ion exchange chromatography is employed to purify alkaloids such as arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, wherein said ion exchange chromatography process is performed based on known principles. For instance, by employing ion exchange 25 chromatography (IEX) in the methods of the present disclosure, molecules possessing the opposite charge as the resin’s ligand will bind tightly to the resin, and molecules having the same charge as the resin’s ligand will flow through the chromatographic column and elute out first during intermediate wash step and is collected as initial wash flow through.
  • IEX ion exchange 25 chromatography
  • the cation exchange chromatography is employed to purify arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, wherein said cation exchange chromatography process is performed based on known principles. For instance, by employing cation exchange chromatography in the methods of the present disclosure, arecoline possessing the opposite charge as the resin binds tightly to the resin and is retained, whereas other molecules having the same charge as the resin flow through the chromatographic column and elute out first.
  • the hydrophobic interaction chromatography is employed to 5 purify polyphenols selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, wherein said hydrophobic interaction chromatography (HIC) process is performed based on known principles. For instance, by employing hydrophobic interaction chromatography in the 10 methods of the present disclosure, separation of polyphenols based on their hydrophobicity takes place.
  • HIC is also a particularly useful separation technique for purifying phytochemicals from areca wash liquid as this technique maintains activity of the desired molecules due to the use of conditions and matrices that operate under less denaturing conditions.
  • the principle of HIC is complementary to ion exchange chromatography.
  • HIC 15 using macroporous polymeric hydrophobic resins the sample molecules containing hydrophobic and hydrophilic regions are applied to an HIC column in a low solvent low salt conditions. The more hydrophobic the phytomolecules, the more solvation it is needed to elute.
  • an increasing salt condition is used to elute samples from the column in order of increasing hydrophobicity or a slight change of polarity.
  • Sample elution may also be 20 assisted by the addition of mild organic modifiers or detergents to the elution buffer.
  • IEX ion exchange chromatography
  • Diaion SK1B from Mitsubishi chemicals (Cation Exchange Resin), or Amberlite 30 IRC 120H from Merck-Supelco, Germany (Cation Exchange Resin), or Amberlite IRA 400
  • the hydrophobic interaction chromatography (HIC) employed in the purification of phytochemicals from areca wash liquid (AWL) in the present disclosure is performed using the following parameters/conditions: a) Hydrophobic Interaction Chromatography:
  • Sample Binding 1.75 CV of Permeate obtained from the membrane fdtration; permeate 30 obtained from microfdtration (pre -fdtration technique); or initial wash flow through obtained from IEX
  • Fraction Collection 12 fractions of 50mL each when the samples were eluted using 20%, 30 40% and 60% Ethanol.
  • the methods for purification of phytochemicals from areca wash liquid (AWL) as described above comprises subjecting the areca wash liquid to a prefiltration step using Whatman filter paper, depth filter, microfiltration, centrifugation, or any combination thereof, before subjecting said areca wash liquid to membrane filtration, chromatography, or the combination of membrane filtration and chromatography.
  • wash liquid ABL
  • ADL wash liquid
  • the above described methods for purification of polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL) is carried out at a temperature ranging from about 18°C to about 68°C.
  • the above described methods for purification of polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL) is carried out at a pressure ranging between 1 bar to 20 bars.
  • the above described methods for purification of polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL) is carried out for a time-period ranging from about 0.5 hours to about 20 hours.
  • the above described methods for purification of polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL) is carried out at a temperature ranging from about 18 °C to about 68 °C at a pressure ranging from 1 bar to 20 bars and for a time-period ranging from about 0.5 hours to about 20 hours.
  • the above described methods for purification of phytochemicals - polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL), results in said phytochemicals having a purity ranging from about 65% to about 100%.
  • the above described methods for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), results in polyphenol having a purity ranging from about 65% to about 100%.
  • the above described methods for purification of ursolic acid (triterpene) or its isomers, from areca wash liquid (AWL) results in ursolic acid having a purity ranging from about 65% to about 100%.
  • the above described methods for purification of catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL), results in catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, or any combination thereof, having a purity of about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%,
  • the above described methods for purification of arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL) results in arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, having a purity ranging from about 65% to about 100%.
  • the above described methods for purification of arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL) results in arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, having a purity of about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%,
  • the above described methods for purification of polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL), results in polyphenols, alkaloid and/or triterpene(s) with an yield ranging from about 1.5% to about 85%.
  • the above described methods for purification of arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL) results in a yield ranging from about 1.5% to about 5.5%.
  • the above described methods for purification of catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL) results in a yield ranging from about 5% to about 85%.
  • the above described methods for purification of ursolic acid from areca wash liquid (AWL) results in ursolic acid yield ranging from about 0.2% to about 5%.
  • the other mentioned compounds described herein, and the isomers of such compounds are also isolated resulting in a yield ranging from about 5% to about 30%.
  • ADL wash liquid
  • Polyphenols such as catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid and resveretrol, and alkaloids such as arecoline
  • ECG epigallocatechin gallate
  • syringic acid coumaric acid and resveretrol
  • alkaloids such as arecoline
  • arecaidine, guvacine and guvacoline are important phytochemical molecules present in higher quantity in the AWL than in the arecanuts that are marketed and exported to foreign countries.
  • EGCGs and catechin are polyphenolic antioxidants whereas, arecoline is a naturally derived free basic alkaloid.
  • Applications of EGCG and catechin are majorly in industries such as Nutraceutical, Cosmetics and Skincare, Pharmaceutical, Food and Beverages, and Consumer electronics. These molecules exhibit properties such as antioxidative property, glucosidase inhibitory property and anti-hypertensive property.
  • Arecoline exhibits parasympathetic and muscarinic properties and is a cholinergic agent.
  • Arecoline is majorly used in sports supplements which help individuals to stay active and energetic and in some special dietary supplements that help in improvising the learning ability and neural activity of people with neural/leaming disorders.
  • Arecoline being an anthelminthic drug also finds its application in animal and pet anthelminthic formulations.
  • the present disclosure provides a method for isolation/purification of the said phytochemicals from an economical raw material - areca wash liquid (AWL).
  • the products obtained from the purification methods described herein is selected from a) an alkaloid product comprising either arecoline, arecaidine, guvacine, guvacoline or any combination thereof; b) a polyphenol product comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), 5 syringic acid, coumaric acid, resveretrol, or any combinations thereof; c) a product comprising a) and b); or d) a product comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid and resveretrol.
  • the method of the present disclosure employs steps including but not limited to membrane processes such as ultrafiltration, microfiltration, depth filtration, filter paper based clarification, or any combination thereof; chromatographic processes such as cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography (using polymeric macroporous hydrophobic resins), other adsorption 15 chromatographic techniques, or any combinations thereof.
  • membrane processes such as ultrafiltration, microfiltration, depth filtration, filter paper based clarification, or any combination thereof
  • chromatographic processes such as cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography (using polymeric macroporous hydrophobic resins), other adsorption 15 chromatographic techniques, or any combinations thereof.
  • the method employs an economical/cost-effective raw material or input (areca wash liquid) for isolating/purifying industrially important phytochemicals (polyphenols and alkaloids),
  • Said raw material (areca wash liquid) has not been employed before for isolating or purifying polyphenols and/or alkaloids.
  • the areca wash liquid is considered majorly a waste material and is discarded after agro- 25 processing of arecanuts;
  • AWL can pose a threat to the environment if discarded into soil. Particularly, it can cause acidification of the soil because of the acidic pH of the material (AWL).
  • the present method employs an alternate source/raw material for isolating polyphenols compared to conventional raw materials or sources (eg. green tea, gambier, acacia). Isolation
  • D. purification method of the present disclosure employs simple and green/environmentally friendly process conditions/parameters and avoids usage of harsh conditions. It is to be understood that the foregoing descriptive matter/embodiments are illustrative of the disclosure and not a limitation. While considerable emphasis has been placed herein on the particular/preferable features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred 5 embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.
  • Fresh areca wash liquid was obtained from various cultivators/farmers in Yellapur, North Canara district of Karnataka. Briefly, said cultivators/farmers prepared the areca wash liquid (AWL) by:
  • the AWL was used for boiling 2-3 batches of fresh whole arecanuts with the addition of 5 extra volume of water. h) The obtained AWL from step g) was thereafter stored in a concrete container or HDPE canisters and was used as a raw material for further experiments.
  • the fresh AWL was transported in PET cans to the R&D premises at Bangalore 10 Bioinnovation Centre (BBC), Bengaluru.
  • the AWL was subjected to Phytochemical analysis (Qualitative analysis) to determine the overall constituents/composition of AWL. Particularly, tests for Alkaloids, Flavonoids, Tannins, Protein and Carbohydrates were performed. Quantitative tests included test for polyphenols, i.e., determination of total polyphenolic content in the AWL by using the standard Folin-Ciocalteu reagent and by 15 measuring the absorbance at 765nm by using gallic acid as standard. From the qualitative analysis of the AWL, the presence of alkaloids, flavonoids, tannins, saponins and minute traces of carbohydrates were detected.
  • the flavonoids consisted of majorly polyphenols belonging to the group of epigallocatechin (EGC), catechin, epicatechin, epigallocatechin gallate (EGCG), syringic acid, epicatechin gallate (EGC) at higher concentrations, and 20 resveratrol, quercetin in minor concentrations.
  • Example 2 Purification of alkaloids and polyphenols by ion exchange chromatography (IEX), membrane filtration, and hydrophobic interaction chromatography (HIC)
  • Example 1 The AWL obtained in Example 1 was used for isolation/purification of the compounds of 25 interest (alkaloids and polyphenols) as follows:
  • Centrifugation - AWL samples were centrifuged at 24,000 rpm for about 20 minutes. For industrial/commercial scale, continuous centrifugation may be employed to handle sample 30 volumes of a large scale.
  • Ion exchange chromatography IEX
  • membrane filtration microfiltration followed by ultrafiltration
  • HIC hydrophobic interaction chromatography
  • Example 3 Purification of alkaloids and polyphenols by membrane filtration, hydrophobic interaction chromatography (HIC) and ion exchange chromatography HEX)
  • Example 2 The pre-filtered AWL of Example 2 was used for isolation/purification of the compounds of 20 interest (alkaloids and polyphenols).
  • the process conditions/parameters of membrane filtration (MF), hydrophobic interaction chromatography (HIC) and ion exchange chromatography (IEX) remain the same as described in Example 2.
  • the samples used in each process vary which is as follows:
  • HIC a.
  • Sample Permeate obtained from ultrafiltration. pH adjusted between 3 to 8.
  • Example 4 Purification of alkaloids and polyphenols by membrane filtration, ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC)
  • IEX ion exchange chromatography
  • HIC hydrophobic interaction chromatography
  • the pre-filtered AWL of Example 2 was used for isolation/purification of the compounds of interest (alkaloids and polyphenols).
  • the process conditions/parameters of membrane 5 filtration (MF), ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC) remain the same as described in Example 2.
  • the samples used in each process vary which is as follows:
  • MF a.
  • Microfiltration Sample: prefiltered AWL (centrifuged at 24,000 rpm for 20 minutes) and 10 diluted with 10% ethanol
  • Ultrafiltration Undiluted permeate obtained from microfiltration
  • IEX a. Sample: Permeate obtained from ultrafiltration. pH adjusted between 3 to 8.
  • Example 5 Purification of alkaloids by membrane filtration and ion exchange chromatography (IEX)
  • Example 2 The pre-filtered AWL obtained in Example 2 was used for isolation/purification of alkaloids.
  • MF membrane filtration
  • IEX ion exchange chromatography
  • Sample Permeate obtained from ultrafiltration. pH adjusted between 3 to 8.
  • Example 2 The pre-filtered AWL obtained in Example 2 was used for isolation/purification of alkaloids.
  • the process conditions/parameters of ion exchange chromatography (IEX) remain the same as described in Example 2.
  • the sample used in this experiment is as follows:
  • the sample may be diluted with Di water or ethanol or IPA if it is too viscous.
  • Example 7 Purification of polyphenols by membrane filtration and hydrophobic interaction chromatography (HIC)
  • Example 15 The pre-filtered AWL obtained in Example 2 was used for isolation/purification of polyphenols.
  • the samples used in these processes were as follows:
  • Sample Permeate obtained from ultrafiltration. pH adjusted between 3 to 8.
  • Example 8 Purification of polyphenols by hydrophobic interaction chromatography (HIC)
  • Example 2 The pre-filtered AWL obtained in Example 2 was used for isolation/purification of 30 polyphenols.
  • the process conditions/parameters of hydrophobic interaction chromatography were used for isolation/purification of 30 polyphenols.
  • HIC Sample: Prefiltered AWL (Centrifuged at 24,000 rpm for about 20 minutes). This step was carried out twice to get rid of any solid impurities that might clog the resins packed during HIC.
  • the sample may be diluted with DI water or ethanol or IPA if it is too viscous.
  • HPLC method was optimized on a Waters Automated HPLC, with quaternary pump, auto sampler, PDA detector using a reverse phase C-18 Column. The following conditions were employed:
  • Mobile phase contained: Acetonitrile (ACN), Isoproyl alcohol(IPA), orthophosphoric acid, Di Water and Methanol [Mobile phase A: 0.1% orthophosphoric acid; Mobile phase B: ACN:IPA (95:5); Di Water and Methanol was used for final column washing].
  • Flow rate 1 ml/min
  • Standard polyphenols (0.25-lmg/mL) such as catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), Epigallocatechin gallate(EGCG), Quercetin, Syringic acid, and Alkaloid: arecoline was run.
  • Sample size was optimized by varying volume of injection and sample dilution. Samples were diluted from lx to lOx and volumes were taken from lOpl to lOOpl to get the optimal peak shapes, resolution and quantification.
  • the chromatograms provided in Figures 8A and 8B show the presence of alkaloid and polyphenols in the AWL. Further, the AWL was subjected to purification by membrane filtration and the final separation of alkaloids (arecoline) and polyphenols such as catechin, epicatechin and epigallocatechin (EGC) were carried out through IEX and HIC respectively.
  • alkaloids arecoline
  • polyphenols such as catechin, epicatechin and epigallocatechin (EGC) were carried out through IEX and HIC respectively.

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Abstract

The instant disclosure is in the field of biochemical sciences, particularly towards bioseparation/purification of phytochemicals. The disclosure relates to method for purification of phytochemicals from areca wash liquid (AWL). More particularly, the present method provides purification of phytochemicals selected from polyphenols, alkaloids, triterpenes, and combinations thereof, from AWL.

Description

“PURIFICATION OF PHYTOCHEMICAL(S) FROM ARECA WASH LIQUID”
TECHNICAL FIELD
The instant disclosure is in the field of biochemical sciences, particularly towards bioseparation/purification of phytochemicals. The disclosure relates to method for purification of phytochemicals from areca wash liquid (AWL) . More particularly, the present method provides purification of phytochemicals selected from alkaloids, polyphenols, triterpenes or any combination thereof, from AWL.
BACKGROUND OF THE DISCLOSURE
Areca plant (Areca catechu Linn.) is a tropical plant primarily grown in parts of Southeast Asia. Betel nut or arecanut has been used as a masticator by humans since the 4th century AD. Arecanut is chewed by nearly 15% of the world population or nearly 1.1 billion people. In different parts of the world, arecanut is consumed in different ways. In India, the arecanut is consumed along with the betel leaves (the leaves of plant related to pepper) along with CaCOs or lime forming a dark red mixture upon mastication. India is a major hotspot for the production of arecanut which accounts to 6.5 million arecanut farmers and dominates worldwide production by producing 57-60% of the total produce (7,45,860 tonnes in 2016). In India, Karnataka produces about 55% of the areca produce (4,16,290 tonnes in 2016) which accounts to nearly 5% of the state’s GDP.
Currently, quests to identify, isolate and produce natural components/ingredients as a replacement for artificial/chemically synthesized ingredients used in the pharmaceutical, cosmeceutical, food and nutraceutical industries is in high demand. While researchers majorly study plants of medicinal value, it is also necessary to investigate, study and understand the capability and potentiality of the molecules obtained from agricultural resources/plants, such as areca plant. Also, it is important for the researchers to know the value of such molecules/components and make use of them in economical and feasible ways.
Areca plant (Areca catechu) is largely focused for its importance/utility of arecanut derived from areca fruit. Given the importance of areca plant as an important agricultural plantation/resource, there is a need for exploring beneficial molecules/components which can be further utilized economically in pharmaceutical, cosmetics, food and nutraceutical industries. The present disclosure addresses said need. BRIEF DESCRIPTION OF FIGURES
Figures 1 to 7 illustrate alternate methods of purifying phytochemicals (alkaloids and/or polyphenols) from areca wash liquid (AWL) according to the present disclosure.
Figures 8 to 14 illustrate chromatograms obtained through analytical HPLC method for AWL, permeate obtained from membrane fdtration, pure fractions obtained from HIC and IEX based on methods described in Examples 2 to 8 and Figures 1 to 7, respectively as follows:
Figure 8: Chromatogram of AWL (raw material) - Figure 8A: Chromatogram of AWL detected at 280nm showing the peaks of the presence of polyphenols [Retention Time (RT) 9.775, 11.697, 12.937, 14.556, 16.847, 20.188 are the peaks for epigallocatechin (EGC), catechin, syringic acid, epicatechin, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) respectively]; Figure 8B: Chromatogram of AWL detected at 216nm showing the peaks of the presence of alkaloids [Retention Time (RT) 2.57 is the arecoline peak].
Figure 9: Chromatogram of method results based on Example 2 and Figure 1 - Figure 9A: Chromatogram of pure fraction (IEX) of Arecoline detected at 216nm (RT 2.719); Figure 9B: Chromatogram of ultrafiltration (membrane filtration) permeate detected at 280nm indicating the presence of polyphenols (RT 9.702, 11.579, 12.859, 14.038, 16.711, 20.086 are the peaks for epigallocatechin (EGC), catechin, syringic acid, epicatechin, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) respectively); Figure 9C: Chromatogram of pure fraction (HIC) of catechin detected at 280nm (RT 12.993); Figure 9D: Chromatogram of pure fraction (HIC) of EGC detected at 280nm (RT 9.639). Figure 9E: Chromatogram of pure fraction (HIC) of EGCG detected at 280nm (RT 16.573).
Figure 10: Chromatogram of method results based on Example 3 and Figure 2 - Figure 10A: Chromatogram of ultrafiltration (membrane filtration) permeate detected at 280nm indicating the presence of polyphenols (RT 9.702, 11.579, 12.859, 14.438, 16.711, 20.086 are the peaks for epigallocatechin (EGC), catechin, syringic acid, epicatechin, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) respectively); Figure 10B: Chromatogram of ultrafiltration (membrane filtration) permeate detected at 216nm indicating the presence of alkaloids (RT 2.803 is the peak for arecoline); Figure 10C: Chromatogram of pure fraction (HIC) of EGC detected at 280nm (RT 9.911); Figure 10D: Chromatogram of pure fraction (HIC) of Catechin detected at 280nm (RT 12.926); Figure 10E: Chromatogram of pure fraction (HIC) of EGCG detected at 280nm (RT 16.898); Figure 10F: Chromatogram of pure fraction (IEX) of Arecoline detected at 216nm (RT 2.714).
Figure 11 : Chromatogram of method results based on Example 4 and Figure 4 - Figure 11 A: Chromatogram of ultrafiltration (membrane filtration) permeate detected at 280nm indicating the presence of polyphenols (RT 9.702, 11.579, 12.859, 14.438, 16.711, 20.086 are the peaks for epigallocatechin (EGC), catechin, syringic acid, epicatechin, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) respectively); Figure 11B: Chromatogram of ultrafiltration (membrane filtration) permeate detected at 216nm indicating the presence of alkaloid (RT 2.803); Figure 11C: Chromatogram of pure fraction (IEX) of Arecoline detected at 216nm (RT 2.708); Figure 11D: Chromatogram of pure fraction (HIC) of Catechin detected at 280nm (RT 12.926); Figure HE: Chromatogram of pure fraction (HIC) of Epicatechin detected at 280nm (RT 14.984); Figure 11F: Chromatogram of pure fraction (HIC) of EGC detected at 280nm (RT 9.785); Figure 11G: Chromatogram of pure fraction (HIC) of EGCG detected at 280nm (RT 16.909).
Figure 12: Chromatogram of method results based on Examples 5 & 6 and Figures 4 & 5. The figure depicts chromatogram of pure fraction (IEX) of Arecoline detected at 216nm (RT 2.745).
Figure 13: Chromatogram of method results based on Example 7 and Figure 6 - Figure 13 A: Chromatogram of ultrafiltration (membrane filtration) detected at 280nm indicating the presence of polyphenols (RT 9.497, 11.017, 12.860 are the peaks for epigallocatechin (EGC), catechin and syringic acid respectively); Figure 13B: Chromatogram of ultrafiltration (membrane filtration) detected at 216nm indicating the presence of arecoline (RT 3.096).
Figure 14: Chromatogram of method results based on Example 8 and Figure 7 - Figure 14A: Chromatogram of pure fraction (HIC) of EGC detected at 280nm (RT 9.852); Figure 14B: Chromatogram of pure fraction (HIC) of Catechin, Epicatechin detected at 280nm (RT 12.912, 14.534); Figure 14C: Chromatogram of pure fraction (HIC) of EGCG detected at 280nm (RT 16.898).
DESCRIPTION OF THE DISCLOSURE
As used in the present disclosure, the terms/phrases “areca wash liquid”, “AWL”, “areca boiled sludge” or “ABL” are used interchangeably and refers to the liquid composition derived by processing raw arecanuts as described herein. Details/characterization of areca wash liquid is further provided in the embodiments described below.
As used in the present disclosure, the terms “arecanut”, “areca kernel”, “areca seed” or “betel nut” are used interchangeably and refers to the seed of the areca plant (Areca catechu) obtained by de-husking areca fruit.
As used in the present disclosure, the terms “whole arecanuts” or “whole nuts” are used interchangeably and refers to the use of whole nuts of the areca fruit. In other words, said whole nuts are not cut/shredded into pieces and are used in their complete form/shape.
As used in the present disclosure, the phrase “at least one of alkaloid, polyphenol and triterpene” refers to the isolation/purification of alkaloid(s) alone; polyphenol(s) alone; triterpene(s) alone; alkaloid(s) and polyphenol(s); alkaloid(s) and triterpene (s); polyphenol(s) and triterpene(s); or all of alkaloid(s), polyphenol(s) and triterpene(s).
The present disclosure aims at addressing the need of identifying and isolating value-added components/phytochemicals from alternate agricultural plants/resources, particularly areca plant.
The present disclosure relates to purification of value-added components/molecules from areca wash liquid (AWL).
The present disclosure particularly relates to a method for purification of phytochemicals from areca wash liquid (AWL), said method comprising subjecting the areca wash liquid (AWL) to membrane filtration, chromatography, or a combination of membrane filtration and chromatography; wherein the phytochemical is at least one of alkaloid, polyphenol and triterpene. In some embodiments, the method comprises purification of alkaloids selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof.
5
In some embodiments, the method comprises purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
10
In some embodiments, the method comprises purification of triterpene which is ursolic acid, isomers of ursolic acid, or a combination thereof.
The areca wash liquid is a liquid composition obtained by processing areca fruit of the areca 15 plant.
In some embodiments, the areca wash liquid refers to the liquid composition obtained by processing raw arecanuts, wherein the raw arecanuts are boiled in water at boiling temperature at atmospheric pressure for about 1 hour to 12 hours, preferably 2 hours to 5 20 hours.
In some embodiments, the areca wash liquid (AWL) refers to the liquid composition obtained by processing ‘whole arecanuts’ without cutting/shredding said whole arecanut into pieces. In some embodiments, the whole arecanut remains intact while processing the whole
25 arecanuts to obtain areca wash liquid (AWL).
In some embodiments, the areca wash liquid refers to the liquid composition obtained by a process comprising: a) dehusking areca fruits to obtain arecanuts; kB)O boiling the arecanuts (whole nuts) in presence of a solvent at a temperature of about 50°C to about 100°C for a time-period ranging from about 1 hour to 12 hours, preferably 1 hour to 5 hours, wherein the solvent is selected from a group comprising water, a polar solvent, or a combination thereof; c) separating the liquid remnant/liquid composition from the arecanuts after step b), wherein said liquid composition is the areca wash liquid.
In some embodiments, the areca wash liquid refers to the liquid composition obtained by a 5 process comprising: a) de-husking areca fruits to obtain arecanuts; b) boiling the arecanuts (whole nuts) in presence of a solvent at a temperature of about 50°C to about 100°C for a time-period ranging from about 1 hour to 12 hours, preferably 1 hour to 5 hours; and d)0 separating the liquid remnant from the arecanuts after step b), d) boiling fresh de-husked arecanuts (whole nuts) in presence of the solvent, the separated liquid remnant of step c), at a temperature of about 50°C to about 100°C for a time-period ranging from about 1 hour to 12 hours, preferably 1 hour to 5 hours, e) separating liquid remnant from the arecanuts after step d), wherein said liquid remnant is the 15 areca wash liquid (AWL), wherein the solvent is selected from a group comprising water, a polar solvent, or a combination thereof, and wherein said step d) and step e) are optionally repeated for about 1 to 5 times.
20 In some embodiments of the above process, the solvent in above step b) or step d) is water.
In some embodiments of the above process, the solvent in step b) or step d) is a polar solvent.
In some embodiments, the solvent in above step b) or step d) is a polar solvent selected from a group comprising ethanol, methanol, ethyl acetate, isopropyl alcohol (IPA) and 25 combinations thereof. In some embodiments, the process of obtaining areca wash liquid comprises adding bases such as lime (calcium hydroxide) in addition to the solvent.
In some embodiments, the boiling of whole arecanuts [above step b) - boiling the whole arecanuts in presence of a solvent] optionally comprises adding a quantity of previously 30 obtained or previously prepared areca wash liquid.
In some embodiments of the above process, the boiling is carried out at atmospheric pressure. In some embodiments, the areca wash liquid refers to the liquid composition obtained by a process comprising: a) harvesting areca fruits from areca plant/tree; b) dehusking areca fruits to obtain arecanuts; c)5 boiling the arecanuts (whole arecanuts) in water at a temperature of about 65 °C to about
100°C and at atmospheric pressure for a time-period ranging from about 2 hours to 5 hours; and d) separating liquid and arecanuts, wherein said liquid is the areca wash liquid.
10 In some embodiments, the areca wash liquid refers to the liquid composition obtained by a process comprising: a) harvesting areca fruits from areca plant/tree; b) de-husking areca fruits to obtain arecanuts; c) boiling the arecanuts (whole nuts) in water at a temperature of about 65 °C to about 100°C 15 and at atmospheric pressure for a time-period ranging from about 2 hours to 5 hours; and a) separating liquid from the arecanuts after step c), to obtain separated liquid and boiled arecanuts, d) boiling fresh de-husked arecanuts (whole nuts) in water and the separated liquid of step d), at a temperature of about 65 °C to about 100°C and at atmospheric pressure for a time -period
20 ranging from about 2 hours to 5 hours; and e) separating liquid from the arecanuts after step e), wherein said liquid is the areca wash liquid (AWL), and wherein said step e) and step f) are optionally repeated for about 1 to 5 times.
25 In some embodiments, the areca wash liquid refers to the liquid composition obtained by a process comprising: a) harvesting areca fruits from areca plant/tree; b) dehusking areca fruits to obtain arecanuts; c) boiling the arecanuts (whole nuts) in presence of a polar solvent selected from a group 30 comprising ethanol, methanol, ethyl acetate and combinations thereof, at a temperature of about 50°C to about 85°C and at atmospheric pressure for a time -period ranging from about 1 hour to 3 hours; and d) separating liquid and arecanuts, wherein said liquid is the areca wash liquid. In some embodiments, the areca wash liquid refers to the liquid composition obtained by a process comprising: a) harvesting areca fruits from areca plant/tree; b) de-husking areca fruits to obtain arecanuts; c)5 boiling the arecanuts (whole nuts) in presence of a polar solvent at a temperature of about
50°C to about 85°C and at atmospheric pressure for a time -period ranging from about 1 hour to 3 hours; and d) separating liquid from the arecanuts after step c), to obtain separated liquid and boiled arecanuts, d)0 boiling fresh de-husked arecanuts (whole nuts) in a polar solvent and the separated liquid of step d), at a temperature of about 50°C to about 85°C and at atmospheric pressure for a timeperiod ranging from about 1 hour to 3 hours, at a temperature of about 65 °C to about 100°C and at atmospheric pressure for a time-period ranging from about 2 hours to 5 hours; and f) separating liquid from the arecanuts after step e), wherein said liquid is the areca wash liquid 15 (AWL), wherein the polar solvent is selected from a group comprising ethanol, methanol, ethyl acetate and combinations thereof, and wherein said step e) and step f) are optionally repeated for about 1 to 5 times.
20 In some embodiments, the areca wash liquid refers to the liquid composition obtained by a method comprising steps of: a) harvesting areca fruits from areca plant/tree; b) dehusking areca fruits by pealing/removing the outer fibrous husk of the areca fruits to obtain arecanuts;
<2)5 boiling the arecanuts (whole nuts) in a vessel or a tank in the presence of a solvent selected from a group comprising water, ethanol, methanol, ethyl acetate and combinations thereof, along with mild addition of a base such as calcium hydroxide at concentration ranging from about 0.5 to 7.5% w/v, at a temperature of about 55°C to about 100°C and at atmospheric pressure for a time-period ranging from about 1 hour to 4 hours, and wherein said boiling 30 optionally comprises adding a quantity of previously obtained areca wash liquid; and d) separating liquid and arecanuts, wherein said liquid is the areca wash liquid.
In some embodiments, the areca wash liquid refers to the liquid composition obtained by a process comprising: a) harvesting areca fruits from areca plant/tree; b) de-husking areca fruits by pealing/removing the outer fibrous husk of the areca fruits to obtain arecanuts; c) boiling the arecanuts (whole nuts) in a vessel or a tank in presence of a solvent along with 5 mild addition of a base at concentration ranging from about 0.5 to 7.5% w/v, at a temperature of about 55°C to about 100°C and at atmospheric pressure for a time-period ranging from about 1 hour to 4 hours, d) separating liquid from the arecanuts after step c), to obtain separated liquid and boiled arecanuts, d)0 boiling fresh de-husked arecanuts (whole nuts) in presence of a solvent and the separated liquid of step d) along with mild addition of a base at concentration ranging from about 0.5 to 7.5% w/v, at a temperature of about 55°C to about 100°C and at atmospheric pressure for a time-period ranging from about 1 hour to 4 hours; and f) separating liquid from the arecanuts after step e), wherein said liquid is the areca wash liquid 15 (AWL), wherein the solvent is selected from a group comprising water, ethanol, methanol, ethyl acetate and combinations thereof, wherein the base is calcium hydroxide, and wherein said step e) and step f) are optionally repeated for about 1 to 5 times.
20
In some embodiments, the above described process of obtaining the areca wash liquid further comprises optionally agitating the vessel or tank comprising arecanuts and solvent during the boiling of the arecanuts, wherein said agitation is for one or more times.
25 In some embodiments, the areca wash liquid refers to the liquid composition essentially comprising: a) polyphenols, b) alkaloids, c) polysaccharide, d) saponins, e) triterpenes, f) steroids, g) minerals, and h) optionally, a residual component selected from a group comprising sugars, proteins, tannins aggregates, fat, impurities and combinations thereof.
30 In some embodiments, the areca wash liquid refers to the liquid composition essentially comprising: a) polyphenols, b) alkaloids, c) saponins, d) triterpenes, e) steroids, f) polysaccharide, g) minerals, and h) a residual component selected from a group comprising sugars, proteins, tannin aggregates, fat, impurities and combinations thereof. In some embodiments, the areca wash liquid essentially comprises: a) polyphenols selected from catechin, epigallocatechin gallate (EGCG), epicatechin isomer of EGCG, isomer of catechin, isomer of epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), syringic acid, resveretrol, quercetin, gallic acid, coumaric acid, leucocyanidins, dimeric and complex cyanidins, luteolin, liquiritigenin, chrysoeriol, isorhamnetin, or any combination thereof; b) alkaloids selected from arecoline, guvacine, arecaidine, guvacoline or any combination thereof; c) triterpenes and steroids selected from ursolic acid, sitosterol, arborinol, cylindrin, femenol, cycloartenol, ergosterol, isomers thereof, or any combination thereof; d) polysaccharide selected from starch, hemicellulose, or a combination thereof; e) minerals selected from potassium, calcium, sodium, phosphorous, iron, or any combination thereof; f) vitamins selected from vitamin C, vitamin B6, or a combination thereof; and g) residual component selected from a group comprising sugars, proteins, tannin aggregates, fat, impurities and combinations thereof, wherein the proteins comprise amino acids selected from proline, tyrosine, phenylalanine, phenylalanine, methionine, arginine, aspartic acid, glutamic acid, lysine, tryptophan, or any combination thereof.
In some embodiments, the areca wash liquid when dehydrated and analyzed essentially comprises polyphenols at a concentration ranging from about 15 wt % to about 70 wt %, alkaloids at a concentration ranging from about 0.5 wt % to about 4.5 wt %, polysaccharide at a concentration ranging from about 12 wt % to about 30 wt %, minerals at a concentration ranging from about 0.2 wt % to about 2 wt %, and residual component at a concentration ranging from about 0.3 wt % to about 1.5 wt %. In embodiments of the present disclosure, the ingredients/components of the areca wash liquid (AWL) add up to 100 wt%.
In some embodiments, the areca wash liquid comprises the following characteristics and composition:
Figure imgf000012_0001
Figure imgf000013_0001
In some embodiments, the areca wash liquid also comprises additional components/molecules (apart from the areca wash liquid composition described above) and said additional components/molecules is based on the composition of arecanut. The arecanut 5 composition comprises polyphenol (45-70 wt %), fat (15-19 wt %), starch (20-25 wt %) and alkaloids (1.5-4.5 wt %). Further characterization of the arecanut composition is as follows:
1. Polyphenols: The polyphenols present in arecanut are mostly flavonols, that include about 12-30% of catechin, about 8-20% epicatechin, about 12-17% of leucocyanidin, the remaining portion being complex flavonoids in varying degrees of polymerization. A series
10 of dimeric, trimeric and tetrameric procyanidins have been isolated from arecanut. These are mainly, epigallocatechin gallate (about 3-15%), catechin gallate (about 2-10%), epicatechin gallate. Quercetin, coumaric acid and gallic acid are also present.
2. Alkaloids: The four major alkaloids present in arecanut are arecoline (about 7.5 to 21.5 mg/g), arecaidine (about 1.5 to 6.2 mg/g), guvacoline (about 2.0 to 5.0 mg/g) and guvacine
15 (about 2.9 to 5.3 mg/g). All these alkaloids are chemically related. Arecoline is a colorless volatile alkaloid.
3. Triterpenes and steroids: 1-6% dry weight of arecanut comprises of these class of molecules such as ursolic acid, femenol, sitosterol, ergosterol, cylindrin and other traces.
4. Fat: Fats consist of about 15 to 19% dry weight of arecanut. Arecanut fatty acid profile 20 comprises about 19.5% lauric acid, about 46.2% myristic acid, about 12.7% palmitic acid, about 1.6% stearic acid, about 0.3% decanoic acid, about 6.2% oleic acid, about 5.4% dodecenoic acid, about 0.3% tetra decanoic acid and about hexadecenoic acid 7.2%.
5. Polysaccharides: Starch is the primary polysaccharide and accounts for about 20 to 25% of polysaccharides.
( 5 Mineral and ash content: The mineral content primarily includes calcium, phosphorus and iron. It also contains traces of Vitamin B6 and Vitamin C. In some embodiments, the concentrations of the components in the areca wash liquid vary based on the types of solvent (water or an alcoholic solvent) employed for obtaining areca wash liquid. For instance, employing solvents such as ethanol or methanol may affect the yield of the components in the areca wash liquid, wherein the yield may increase by 1.5 to 2 times when compared to the areca wash liquid obtained by employing water as a solvent.
The processing of arecanut varies from region to region and country to country. An important agro-processing method is the boiling of the arecanut which is traditionally followed in the western ghats and malenadu (rainfall) parts of Kamataka/Kerala (India) for obtaining the highest quality ‘red variety’ of the arecanuts. The by-product of this processing method is a red coloured areca wash liquid (AWL) also referred to as Chogaru (in Kannada) or Kalipak (in Malayalam) in the native Indian languages, wherein said AWL is rich in industrially important natural flavonoids, polyphenols, tannins and alkaloids.
Thus, the areca wash liquid employed in the present disclosure is the liquid composition obtained by the process described above and/or the liquid composition comprising the composition as described above. Any variations in the process of obtaining areca wash liquid and/or the composition areca wash liquid as described herein is well within the purview of a skilled person in the art. Particularly, the areca wash liquid may also be obtained by any other areca fruit processing method(s) as long as the obtained areca wash liquid comprises phenols and/or alkaloids. More particularly, the areca wash liquid may also be obtained by any other areca fruit processing method(s) as long as the obtained areca wash liquid comprises one or more of arecoline, arecaidine, guvacine, guvacoline, epigallocatechin gallate (EGCG), catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), syringic acid, coumaric acid, resveretrol, ursolic acid, and/or isomers of said compounds.
The present disclosure thus relates to a method for purification of phytochemicals from areca wash liquid (AWL).
In some embodiments, the present disclosure describes a method for purification of phytochemicals selected from a group comprising polyphenols, alkaloids, triterpenes, and combinations thereof, from areca wash liquid (AWL). In some embodiments, the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline and combinations thereof, from areca wash liquid (AWL).
In some embodiments, the present disclosure describes a method for purification of polyphenols selected from catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL).
In some embodiments, the present disclosure describes a method for purification of triterpenes selected from a group comprising ursolic acid, its isomers, and a combination thereof, from areca wash liquid (AWL).
In some embodiments, the present disclosure relates to a method for purification of alkaloids and polyphenols as described above.
In some embodiments, the present disclosure describes a method for purification of phytochemicals selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from AWL.
In some embodiments, the present disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), said method comprising subjecting the areca wash liquid to membrane filtration, chromatography, or a combination of membrane filtration and chromatography. In some embodiments, the method additionally comprises employing polar and non-polar solvents aiding the purification/separation processes.
In some embodiments, the present disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), said method comprising subjecting the areca wash liquid to membrane filtration and chromatography in any order.
In some embodiments, the present disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), said method comprising subjecting the areca wash liquid to membrane filtration, chromatography, or a combination of membrane filtration and chromatography in any order, wherein the phytochemical is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
In some embodiments, the chromatography is ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), or a combination of both.
In some embodiments, the ion exchange chromatography (IEX) is cation exchange chromatography or anion exchange chromatography. In some embodiments, the cation exchange chromatography purifies at least one alkaloid selected from arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination of alkaloid compounds thereof. However, in some embodiments, if the cation exchange chromatography doesn’t result in efficient purification, anion exchange chromatography can be used as a standalone or additional chromatographic technique for the purification of same alkaloids.
In some embodiments, the hydrophobic interaction chromatography (HIC) purifies at least one polyphenol selected from the group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof; and/or triterpenes (ursolic acid or isomers thereof). In some embodiments, the hydrophobic interaction chromatography employed here could be understood as a chromatography which works by adsorption of molecules such as the above described polyphenols and other such molecules, and desorption and elution based on polarity, hydrophobicity and optionally accompanied by a charge/ionic interaction.
In some embodiments, the membrane filtration is selected from cross flow filtration (CFF), depth filtration, or a combination thereof. In some embodiments, the membrane filtration employed is ultrafiltration and/or microfiltration, based on the principles/mechanism of cross flow filtration (CFF). In some embodiments, the membrane filtration separates large molecules selected from at least one of sugars, proteins, tannin aggregates, fats, other high molecular weight components, impurities, and combinations thereof.
5 In some embodiments, the areca wash liquid is optionally subjected to pre-filtration using Whatman filter paper, depth filter, centrifugation, microfiltration, ultrafiltration, continuous centrifugation or any combination thereof, before subjecting the areca wash liquid to membrane filtration, chromatography, or the combination of membrane filtration and chromatography .
10
In some embodiments, the present disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), said method comprising subjecting the AWL to at least one of membrane filtration, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography (HIC), or any 15 combination of membrane filtration, cation exchange chromatography, anion exchange chromatography and hydrophobic interaction chromatography (HIC) in any order, wherein the phytochemical is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers 20 of said compounds, and combinations thereof.
In some embodiments, the present disclosure describes a method for purification of alkaloids and polyphenols from areca wash liquid (AWL), wherein the alkaloid is selected from the group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, 25 and combinations thereof, and the polyphenol is selected from the group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i. subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify alkaloid; 30 and ii. performing membrane filtration, followed by hydrophobic interaction chromatography
(HIC) to purify the polyphenol. In some embodiments, the present disclosure describes a method for purification of alkaloids and polyphenols from areca wash liquid (AWL), wherein the alkaloid is selected from the group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, and the polyphenol is selected from the group comprising 5 catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i. subjecting the areca wash liquid to ion exchange chromatography (IEX) to obtain initial wash flow through liquid (IWFT) and eluate, wherein the eluate comprises the alkaloid; and iiO subjecting the initial wash flow through liquid (IWFT) to membrane filtration, followed by hydrophobic interaction chromatography (HIC) to obtain an eluate comprising the polyphenol.
In some embodiments, the method for purification of phytochemicals selected from a group 15 comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds and combinations thereof, from areca wash liquid (AWL) is illustrated in Figure 1. The process determining/sensitive step according to the purification method of Figure 1 is the ion 20 exchange chromatography (IEX).
In some embodiments, the present disclosure describes a method for purification of alkaloids and polyphenols comprising steps of: i) subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the 25 alkaloids, wherein the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, tB)O loading areca wash liquid (sample) onto the column and binding at a pH between 3.5 to 10.5, c) intermediate wash of the column with the equilibration buffer of step a) to obtain initial wash flow through liquid (IWFT) comprising unbound molecules, and d) elution of the column using an elution buffer of pH between 2 to 11 to replace the alkaloids with ionic groups, and obtaining an eluate comprising the alkaloid; and ii) subjecting the initial wash flow through liquid (IWFT) obtained from the intermediate wash of the ion exchange chromatography [step c)] to membrane filtration, followed by hydrophobic interaction chromatography (HIC) to purify the polyphenols, wherein the
5 membrane filtration comprises steps of: a) equilibrating a polymeric membrane with solvent (mobile phase), b) applying the initial wash flow through liquid (IWFT) (sample) onto the membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, and c) washing and re-equilibrating the membrane,
10 and wherein the hydrophobic interaction chromatography (HIC) comprises steps of: a) equilibrating a HIC column packed with macro porous polymeric resin using an equilibration buffer having a pH of 3.5 to 10.5, b) application of the permeate (sample) and intermediate wash using the equilibration buffer of step a), and d)5 elution using an elution buffer of pH of 2.5 to 10.5 to obtain an eluate comprising polyphenol; wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof; and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin 20 (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
In some embodiments, the present disclosure describes a method for purification of alkaloids and polyphenols from areca wash liquid (AWL), wherein the alkaloid is selected 25 from the group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, and the polyphenol is selected from the group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i30 subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the alkaloid, and obtaining initial wash flow through liquid (IWFT) and an eluate, wherein the ion exchange chromatography comprises steps of: a) equilibration with suitable buffer of pH between about 3.5-10.5, b) binding at a pH between about 3.5-10.5, c) intermediate wash with the same equilibration pH buffer to obtain initial wash flow through liquid (IWFT) comprising unbound molecules, d) elution using a stronger (higher conductivity) buffer of pH between about 2-11 to replace the alkaloids with ionic groups, and obtaining an eluate comprising the alkaloid; and ii.5 subjecting initial wash flow through liquid (IWFT) obtained from the intermediate wash of the ion exchange chromatography [step c)] to membrane filtration, followed by hydrophobic interaction chromatography (HIC) to purify the polyphenol, wherein the membrane filtration comprises steps of: a) equilibrating a membrane of about 20000kD-lkD or lesser m.w.c.o. (polymeric make such
10 as PES or PVDF either in the cassette or in the hollow fibre type) with suitable mobile phase of suitable solvent strength, conductivity and pH, b) applying the initial wash flow through liquid (IWFT) (sample) at an optimized permeate flow and transmembrane pressure (TMP) leading to separation, wherein the permeate being the component of interest and the retentate comprising of impurities, d)5 washing and re-equilibration of the membrane; and the hydrophobic interaction chromatography employs macro porous polymeric resins, and comprises steps of: a) equilibration using mobile phase of suitable ionic strength, solvent strength of about 15% to 100% of polar solvent and a pH of 3.5 - 10.5,
12)0 application of permeate (sample) and intermediate wash using the equilibration mobile phase, and c) elution at a suitable solvent (polar) strength of about 20%-100% at a suitable pH of about 2.5-10.5, to obtain an eluate comprising the polyphenol.
25 In some embodiments, the present disclosure describes a method for purification of alkaloids and polyphenols from areca wash liquid (AWL), wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate
30 (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i. subjecting the areca wash liquid to membrane filtration followed by hydrophobic interaction chromatography (HIC) to purify the polyphenol; and ii. performing ion exchange chromatography (IEX) to purify the alkaloid. In some embodiments, the present disclosure describes a method for purification of alkaloids and polyphenols from areca wash liquid (AWL), wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, 5 and combinations thereof, and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i. subjecting the areca wash liquid to membrane filtration followed by hydrophobic interaction 10 chromatography (HIC) using a macroporous polymeric hydrophobic resin to obtain initial wash flow through liquid (IWFT) and eluate, wherein the eluate comprises the polyphenol; and ii. subjecting the initial wash flow through liquid (IWFT) to ion exchange chromatography (IEX) to obtain an eluate comprising the alkaloid.
15
In some embodiments, the present disclosure describes a method for purification of alkaloids and polyphenols comprising steps of: i) subjecting the areca wash liquid to membrane filtration followed by hydrophobic interaction chromatography (HIC) to purify the polyphenols, wherein the membrane filtration 20 comprises steps of: a) equilibrating a polymeric membrane with solvent (mobile phase), b) applying the areca wash liquid (sample) onto the membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, c) washing and re-equilibrating the membrane,
25 and wherein the hydrophobic interaction chromatography (HIC) comprises steps of: a) equilibrating a HIC column packed with macro porous polymeric resin using an equilibration buffer having a pH of 3.5 to 10.5, b) application of the permeate (sample) onto the HIC column, and intermediate wash using the equilibration buffer of step a) to obtain initial wash flow through liquid (IWFT) comprising
30 unbound molecules, and c) elution using an elution buffer of pH of 2.5 to 10.5 and obtaining an eluate comprising the polyphenol, and ii) subjecting the IWFT obtained from the intermediate wash of HIC [step b)] to ion exchange chromatography (IEX) to purify the alkaloids, wherein the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration 5 buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, b) loading the IWFT (sample) onto the column ,and binding at a pH between 3.5 to 10.5, c) intermediate wash of the column with the equilibration buffer of step a), and dl)0 elution using an elution buffer of pH between 2 to 11 to replace the alkaloids with ionic groups, and obtaining an eluate comprising the alkaloid; wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof; and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin 15 (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
In some embodiments, the method for purification of phytochemicals selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, 20 epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL) is illustrated in Figure 2. The process determining/sensitive step according to the purification method of Figure 2 is the hydrophobic interaction chromatography (HIC). In some embodiments, the present 25 disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), wherein the phytochemical is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i30 subjecting the areca wash liquid to membrane filtration followed by hydrophobic interaction chromatography (HIC) to purify catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, or combinations thereof, wherein the membrane filtration comprises steps of: a) equilibrating a membrane of about 20000kD-lkD or lesser m.w.c.o. (polymeric make such as PES or PVDF either in the cassette or in the hollow fibre type) with suitable mobile phase of suitable solvent strength, conductivity and pH, b) applying sample (areca wash liquid) at an optimized permeate flow and TMP leading to
5 separation, wherein the permeate being the component of interest and the retentate comprising of impurities, c) washing and re-equilibration of the membrane, wherein the hydrophobic interaction chromatography employs macro porous polymeric resins, and comprises steps of: aO)0 equilibration using mobile phase of suitable ionic strength, solvent strength of about 15% to 100% of polar solvent and a pH of 3.5 - 10.5, b) application of the permeate (sample), and intermediate wash using the equilibration mobile phase to obtain initial wash flow through liquid (IWFT) comprising unbound molecules, and c) elution at a suitable solvent (polar) strength of about 20%-100% at a suitable pH of about 15 2.5-10.5 to obtain an eluate comprising the polyphenol; and ii. subjecting the IWFT to ion exchange chromatography (IEX) to purify arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or combinations thereof, wherein the ion exchange chromatography comprises steps of: a) equilibration with suitable buffer pH between about 3.5-10.5 pH,
12)0 binding at a suitable optimized pH between about 3.5-10.5; c) intermediate wash with the same optimized equilibration pH buffer d) elution using a stronger (higher conductivity) buffer of pH between about 2-11 to replace the alkaloids with ionic groups.
25 In some embodiments, the present disclosure describes a method for purification of alkaloid and polyphenol from areca wash liquid (AWL), wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate
30 (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i. subjecting the areca wash liquid to membrane filtration followed by ion exchange chromatography (IEX) to purify the alkaloid; and ii. performing hydrophobic interaction chromatography (HIC) to purify the polyphenol. In some embodiments, the present disclosure describes a method for purification of alkaloid and polyphenol from areca wash liquid (AWL), wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, 5 and combinations thereof, and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i. subjecting the areca wash liquid to membrane filtration followed by ion exchange
10 chromatography (IEX) to obtain initial wash flow through liquid (IWFT) and eluate, wherein the eluate comprises the alkaloid; and ii. subjecting the IWFT to hydrophobic interaction chromatography (HIC) to obtain an eluate comprising polyphenol.
15 In some embodiments, the present disclosure describes a method for purification of alkaloids and polyphenols comprising steps of: i) subjecting the areca wash liquid to membrane filtration followed by ion exchange chromatography (IEX) to purify the alkaloids, wherein the membrane filtration comprises steps of:
£00 equilibrating a polymeric membrane with solvent (mobile phase), b) applying the areca wash liquid (sample) onto the membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, c) washing and re-equilibrating the membrane, and the ion exchange chromatography comprises steps of:
£05 equilibrating a cation exchange column or an anion exchange column with an equilibration buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, b) loading the permeate (sample) onto the column and binding at a pH between 3.5 to 10.5,
(00 intermediate wash of the column with the equilibration buffer of step a) to obtain initial wash flow through liquid (IWFT) comprising unbound molecules, d) elution of the column using an elution buffer of pH between 2 to 11 to replace the alkaloids with ionic groups, and obtaining an eluate comprising the alkaloid; and ii) subjecting the initial wash flow through liquid (IWFT) obtained from the intermediate wash of the ion exchange chromatography [step c)] to hydrophobic interaction chromatography (HIC) to purify the polyphenols, wherein the hydrophobic interaction chromatography (HIC) comprises steps of: a)5 equilibrating a HIC column packed with macro porous polymeric resin using an equilibration buffer having a pH of 3.5 to 10.5, b) application of the IWFT (sample) onto the HIC column, and intermediate wash using the equilibration buffer of step a), and c) elution of the HIC column using an elution buffer of pH of 2.5 to 10.5 to obtain an eluate 10 comprising polyphenol; wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof; and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric 15 acid, resveretrol, isomers of said compounds, and combinations thereof.
In some embodiments, the method for purification of phytochemicals selected from the group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), 20 syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL) is illustrated in Figure 3. In some embodiments, the present disclosure describes a method for purification of phytochemicals from areca wash liquid (AWL), wherein the phytochemical is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), epicatechin 25 gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, said method comprising: i. subjecting the areca wash liquid to membrane filtration followed by ion exchange chromatography (IEX) to purify arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, wherein the membrane filtration comprises steps 30 of: a) equilibrating a membrane of about 20000kD-lkD or lesser m.w.c.o. (polymeric make such as PES or PVDF either in the cassette or in the hollow fibre type) with suitable mobile phase of suitable solvent strength, conductivity and pH, b) applying areca wash liquid (sample) at an optimized permeate flow and TMP leading to separation, wherein the permeate being the component of interest and the retentate comprising of impurities, c) washing and re-equilibration of the membrane,
5 wherein the ion exchange chromatography comprises steps of: a) equilibration with suitable buffer of pH between about 3.5-10.5, b) loading the permeate (sample) and binding at a suitable optimized pH between about 3.5- 10.5, c) intermediate wash with the same equilibration buffer to obtain initial wash flow through 10 liquid (IWFT) comprising unbound molecules, d) elution using a stronger (higher conductivity) buffer of pH between about 2-11 to replace the alkaloids with ionic groups, to obtain an eluate comprising the alkaloid; and ii. subjecting the IWFT obtained from the intermediate wash of the ion exchange
15 chromatography [step c)] to hydrophobic interaction chromatography (HIC) using macro porous polymeric resins comprises to purify catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, or any combination thereof, wherein the hydrophobic interaction chromatography comprises steps of:
£ 0 equilibration using mobile phase of suitable ionic strength, solvent strength of about 15% to 75% of polar solvent and a pH of 3.5 - 10.5,... b) application of the IWFT (sample), and intermediate wash using the equilibration mobile phase, and c) elution at a suitable solvent (polar) strength of about 20%-100% at a suitable pH of about 25 2.5-10.5 to obtain an eluate comprising polyphenol.
In some embodiments, the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), comprising: i30 subjecting the areca wash liquid to membrane filtration; and ii. performing ion exchange chromatography (IEX) to purify the alkaloid. In some embodiments, the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), comprising: i. subjecting the areca wash liquid to membrane filtration and obtaining a permeate; and ii.5 subjecting the permeate to ion exchange chromatography (IEX), to obtain an eluate comprising alkaloid.
In some embodiments, the present disclosure describes a method for purification of alkaloid comprising steps of: i) 10 subjecting the areca wash liquid to membrane filtration and obtaining a permeate, wherein the membrane filtration comprises steps of: a) equilibrating a polymeric membrane with solvent (mobile phase), b) applying the areca wash liquid (sample) onto the membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, and d)5 washing and re-equilibrating the membrane, and ii) subjecting the permeate to ion exchange chromatography (IEX) to purify the alkaloids, wherein the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration
20 buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, b) loading the permeate (sample) onto the column and binding at a pH between 3.5 to 10.5, c) intermediate wash of the column with the equilibration buffer of step a) to obtain initial wash 25 flow through liquid (IWFT) comprising unbound molecules, and d) elution of the column using an elution buffer of pH between 2 to 11 to replace the alkaloids with ionic groups, and obtaining an eluate comprising the alkaloid; wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof.
30
In some embodiments, the method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL) is illustrated in Figure 4. In some embodiments, the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), comprising: i. subjecting the areca wash liquid to membrane fdtration and obtaining a permeate, wherein the membrane fdtration comprises steps of: a)5 equilibrating a membrane of about 20000kD-lkD or lesser m.w.c.o. (polymeric make such as PES or PVDF either in the cassette or in the hollow fibre type) with suitable mobile phase of suitable solvent strength, conductivity and pH, b) applying areca wash liquid (sample) at an optimized permeate flow and TMP leading to separation, wherein the permeate being the component of interest and the retentate
10 comprising of impurities, c) washing and re-equilibration of the membrane; and ii. subjecting the permeate to ion exchange chromatography (IEX), to purify the arecoline or its isomer, wherein the ion exchange chromatography comprises steps of: a) equilibration with suitable buffer of pH between about 3.5-10.5, ti)5 loading the permeate (sample) and binding at a suitable optimized pH between about 3.5- 10.5, c) intermediate wash with the same equilibration buffer to obtain initial wash flow through liquid (IWFT) comprising unbound molecules, and d) elution using a stronger (higher conductivity) buffer of pH between about 2-11 to replace 20 the alkaloids with ionic groups, to obtain an eluate comprising the alkaloid.
In some embodiments, the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method 25 comprising subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the alkaloid.
In some embodiments, the present disclosure describes a method for purification of alkaloid selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of 30 said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising subjecting the areca wash liquid to ion exchange chromatography (IEX) to obtain an eluate comprising the alkaloid. In some embodiments, the present disclosure describes a method for purification of alkaloids comprising subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the alkaloids, wherein the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration 5 buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, b) loading the areca wash liquid (sample) onto the column and binding at a pH between 3.5 to 10.5,
£(J intermediate wash of the column with the equilibration buffer of step a), and d) elution of the column using an elution buffer of pH between 2 to 11 to replace the alkaloids with ionic groups, and obtaining an eluate comprising the alkaloid; wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof.
15
In some embodiments, the method for purification of arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL) is illustrated in Figure 5. In some embodiments, the present disclosure describes a method for purification of alkaloid selected from arecoline, arecaidine, guvacine, 20 guvacoline, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the alkaloid selected from arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof, wherein the ion exchange chromatography comprises steps of:
£ 5 equilibration of the column with suitable buffer of pH between about 3.5-10.5, b) binding at a suitable optimized pH between about 3.5-10.5, c) intermediate wash of the column with the same optimized equilibration pH buffer, and d) elution of the column using a stronger (higher conductivity) buffer of pH between about 2-
11 to replace the alkaloids with ionic groups, and obtaining an eluate comprising the 30 alkaloid.
In some embodiments, the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising: i. subjecting the areca wash liquid to membrane fdtration; and ii. performing hydrophobic interaction chromatography (HIC) to purify the polyphenol.
5
In some embodiments, the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid
10 (AWL), the method comprising: i. subjecting the areca wash liquid to membrane filtration and obtaining a permeate; and ii. subjecting the permeate to hydrophobic interaction chromatography (HIC) to obtain an eluate comprising the polyphenol.
15 In some embodiments, the present disclosure describes a method for purification of polyphenol from areca wash liquid (AWL), the method comprising steps of: i) subjecting the areca wash liquid to membrane filtration and obtaining a permeate, wherein the membrane filtration comprises steps of: a) equilibrating a polymeric membrane with solvent (mobile phase),
12)0 applying the areca wash liquid (sample) onto the membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, and c) washing and re-equilibrating the membrane, and ii) subjecting the permeate to hydrophobic interaction chromatography (HIC) to purify the
25 polyphenols, wherein the hydrophobic interaction chromatography (HIC) comprises steps of: a) equilibrating a HIC column packed with macro porous polymeric resin using an equilibration buffer having a pH of 3.5 to 10.5, b) application of the permeate (sample) onto the column and intermediate wash of the column
30 using the equilibration buffer of step a), and c) elution of the column using an elution buffer of pH of 2.5 to 10.5 to obtain an eluate comprising polyphenol; wherein the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
In some embodiments, the method for purification of polyphenol selected from a group
5 comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL) is illustrated in Figure 6. In some embodiments, the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), 10 epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising: i. subjecting the areca wash liquid to membrane filtration and obtaining a permeate, wherein the membrane filtration comprises steps of: )5 equilibrating a membrane of about 20000kD-lkD or lesser m.w.c.o. (polymeric make such as PES or PVDF either in the cassette or in the hollow fibre type) with suitable mobile phase of suitable solvent strength, conductivity and pH, b) applying areca wash liquid (sample) at an optimized permeate flow and TMP leading to separation, wherein the permeate being the component of interest and the retentate
20 comprising of impurities, and c) washing and re-equilibration of the membrane; and ii. subjecting the permeate to hydrophobic interaction chromatography (HIC) using macroporous hydrophobic polymeric resins, to purify the polyphenol, wherein the hydrophobic interaction chromatography comprises steps of:
£ 5 equilibration using mobile phase of suitable ionic strength, solvent strength of about 15% to 100% of polar solvent and a pH of 3.5 - 10.5, b) application of sample (permeate), and intermediate wash using the equilibration mobile phase, and c) elution at a suitable solvent (polar) strength of about 20%-100% at a suitable pH of about 30 2.5-10.5, to obtain an eluate comprising the polyphenol.
In some embodiments, the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising subjecting the areca wash liquid to hydrophobic interaction chromatography (HIC) to purify the polyphenol.
5 In some embodiments, the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising subjecting the areca wash liquid to hydrophobic interaction
10 chromatography (HIC) to obtain an eluate comprising the polyphenol.
In some embodiments, the present disclosure describes a method for purification of polyphenol from areca wash liquid (AWL) comprises subjecting the areca wash liquid to hydrophobic 15 interaction chromatography (HIC) to purify the polyphenols, wherein the HIC comprises steps of: a) equilibrating a HIC column packed with macro porous polymeric resin using an equilibration buffer having a pH of 3.5 to 10.5, b) application of the areca wash liquid (sample) on to the column, and intermediate wash of the 20 column using the equilibration buffer of step a), and c) elution of the column using an elution buffer of pH of 2.5 to 10.5 to obtain an eluate comprising the polyphenol; wherein the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), 25 syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
In some embodiments, the method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), 30 epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL) is illustrated in Figure 7. In some embodiments, the present disclosure describes a method for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), the method comprising subjecting the areca wash liquid to hydrophobic interaction chromatography (HIC) to purify the polyphenol, wherein the hydrophobic interaction chromatography employs macro porous polymeric resin(s), and comprises steps of: a)5 equilibration of the column using mobile phase of suitable ionic strength, solvent strength of about 15% to 100% of polar solvent and a pH of 3.5 - 10.5, b) application of areca wash liquid (sample) on to the column, and intermediate wash of the column using the equilibration mobile phase, and c) elution of the column at a suitable solvent (polar) strength of about 20%-100% at a suitable 10 pH of about 2.5-10.5 to obtain an eluate comprising polyphenol.
In some embodiments, the membrane fdtration comprises microfdtration, ultrafdtration or a combination thereof. In some embodiments, the membrane fdtration comprises employing membrane fdters having pore size ranging from about 20000kD to IkD. In some 15 embodiments, the membrane fdtration is employed for separating molecules having a high molecular weight. In some embodiments, the membrane fdtration is employed for separating molecules having a molecular weight ranging from about 1 kDato about 20000 kDa. In some embodiments, the membrane fdtration separates sugars, proteins, fats, tannin aggregates, high molecular weight compounds, impurities, and combinations thereof. In some 20 embodiments, the membrane fdtration is cross flow fdtration (CFF) such as ultrafdtration
(UF) or microfdtration (MF) or both, employed either in the form of cassettes or hollow fibre fdtration systems, or combinations thereof. In some embodiments, the membrane fdtration is accompanied by depth fdtration to ease other separation and fdtration processes.
25 In some embodiments, the crossflow fdtration (CFF) is a membrane fdtration technique in which the starting solution/sample passes tangentially along the surface of the filter. A pressure difference across the filter drives components that are smaller than the pores through the filter. Components larger than the filter pores are retained and pass along the membrane surface.
30
In some embodiments, the membrane fdtration technique employed in the purification of phytochemicals from areca wash liquid (AWL) in the present disclosure is microfdtration and ultrafdtration. In preferred embodiments, the membrane fdtration technique employed in the purification of phytochemicals from areca wash liquid (AWL) in the present disclosure is performed using the following parameters/conditions:
5 Microfiltration:
0.45 p polyethersulfone (PES) Membrane (Sartorius), Hydrosart® (Cellulose based) membrane (Sartorius), polysulfone (PS) membrane, polyvinylidene fluoride (PVDF) membrane, nylon membrane, or polytetrafluoroethylene (PTFE) membrane
Ethanol, water, methanol, IPA or any combination thereof, for dilution of AWL
-10 Transmembrane pressure (TMP) of 1-1.5 bar
- Time period of about 6 hours to 12 hours for processing diluted AWL
Ultrafiltration:
IKDa polyethersulfone (PES) Membrane (Sartorius), Hydrosart® (Cellulose based) 15 membrane (Sartorius), polysulfone (PS) membrane, polyvinylidene fluoride (PVDF) membrane, nylon membrane, or polytetrafluoroethylene (PTFE) membrane
Sample - Permeate obtained from microfiltration
- TMP of 1-2 bar
- Time period of about 48 hours to 96 hours for processing the permeate
20
In some embodiments, the ion exchange chromatography (IEX) is employed to purify alkaloids such as arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, wherein said ion exchange chromatography process is performed based on known principles. For instance, by employing ion exchange 25 chromatography (IEX) in the methods of the present disclosure, molecules possessing the opposite charge as the resin’s ligand will bind tightly to the resin, and molecules having the same charge as the resin’s ligand will flow through the chromatographic column and elute out first during intermediate wash step and is collected as initial wash flow through.
30 In some embodiments, the cation exchange chromatography is employed to purify arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, wherein said cation exchange chromatography process is performed based on known principles. For instance, by employing cation exchange chromatography in the methods of the present disclosure, arecoline possessing the opposite charge as the resin binds tightly to the resin and is retained, whereas other molecules having the same charge as the resin flow through the chromatographic column and elute out first.
In some embodiments, the hydrophobic interaction chromatography (HIC) is employed to 5 purify polyphenols selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, wherein said hydrophobic interaction chromatography (HIC) process is performed based on known principles. For instance, by employing hydrophobic interaction chromatography in the 10 methods of the present disclosure, separation of polyphenols based on their hydrophobicity takes place. HIC is also a particularly useful separation technique for purifying phytochemicals from areca wash liquid as this technique maintains activity of the desired molecules due to the use of conditions and matrices that operate under less denaturing conditions. The principle of HIC is complementary to ion exchange chromatography. In HIC 15 using macroporous polymeric hydrophobic resins, the sample molecules containing hydrophobic and hydrophilic regions are applied to an HIC column in a low solvent low salt conditions. The more hydrophobic the phytomolecules, the more solvation it is needed to elute. Usually, an increasing salt condition is used to elute samples from the column in order of increasing hydrophobicity or a slight change of polarity. Sample elution may also be 20 assisted by the addition of mild organic modifiers or detergents to the elution buffer.
In preferred embodiments, the ion exchange chromatography (IEX) employed in the purification of phytochemicals from areca wash liquid (AWL) in the present disclosure is performed using the following parameters/conditions:
25
IEX:
Column used: Vantage Glass column L- 16 x 500mm, Merck Millipore, any other glass column, Steel column, Polypropylene (PP) column, or HDPE column.
- Resin used: Diaion SK1B from Mitsubishi chemicals (Cation Exchange Resin), or Amberlite 30 IRC 120H from Merck-Supelco, Germany (Cation Exchange Resin), or Amberlite IRA 400
Cl- from Merck-Supelco, Germany (Anion Exchange Resin), or Diaion PAF308L from Mitsubishi chemicals (Anion Exchange Resin).
- Bed packed: 30 cms Sample: AWL prefiltered (centrifuged at about 20,000 rpm to 30,000 rpm for about 10 minutes to 1 hour); or Initial Wash Flow Through obtained from HIC; or permeate obtained from membrane filtration; or permeate obtained from microfiltration (pre -filtration technique)
- 5 Equilibration Buffers: Deionised (Di) Water (pH 3 to 8), HC1 (pH 3 to 6)
- Elution Buffers: 1-8% NaCl, 1-5% NaOH, 1- 5% NH40H
Column Regeneration: 3 to 5% HC1
Sequential Steps: a. Column Equilibration: 2CV (Column Volume) with equilibration buffer
HO Sample Loading: 1.5-2CV with sample conditioned with pH 3 to 8 c. Intermediate Washing: Di Water pH (3 to 8) 2CV d. Elution: Gradient elution or Step Elution using the elution Buffers: 6-8CV e. Final wash or Column regeneration: 2CV
15 In some embodiments, the hydrophobic interaction chromatography (HIC) employed in the purification of phytochemicals from areca wash liquid (AWL) in the present disclosure is performed using the following parameters/conditions: a) Hydrophobic Interaction Chromatography:
-20 Resin Used (Hydrophobic adsorbent): Sepabeeds SP700 from Mitsubishi Chemicals, or Sepabeads SP 825L from Mitsubishi Chemicals, or Amberlite XAD 2 from Merck - Supelco, or Amberlite XAD 7HP from Merck - Supelco
Column Used: Glass Column Vantage L- 22 x 500mm, Merck Millipore, any other glass columns, Steel column, Polypropylene (PP) column, or HD PE column
-25 Column Packed Height: 30 cms to 100 cms
Column Volume (CV): 100 to 200 mb
Column Max Pressure: 1 to 20 Mpa
Solvents Used: Mobile A: Di Water
Mobile B: 85% Iso Propyl Alcohol, ethanol (20% to 100%), or IPA (20% 30 to 80%)
Sequential Steps:
I. Equilibration: 2CV with Di water (pH 3.5 to 8) II. Sample Binding: 1.75 CV of permeate obtained from membrane fdtration; permeate obtained from microfdtration (pre -fdtration technique); or initial wash flow through obtained from IEX
III. Initial Wash: 1.75 CV with Di water (Initial Wash Flow Through)
IV. 5 Elution: Step Elution i. 2CV with 20% IPA ii. 2CV with 40% IPA iii. 2CV with 60% IPA
V. Final Washing: 1.5CV with Di water
10 Fraction Collection: 12 fractions of 50mL each when the samples are eluted using 20%, 40% and 60% IPA. b) Hydrophobic Interaction Chromatography (pH 3,5)
- Resin Used: Sepabeeds SP700 from Mitsubishi Chemicals, or Sepabeads SP 825L from
15 Mitsubishi Chemicals, or Amberlite XAD 2 from Merck - Supelco, or Amberlite XAD 7HP from Merck - Supelco
Column Used: Glass Column Vantage L- 22 x 600mm, Merck Millipore, any other glass columns, Steel column, Polypropylene (PP) column, or HD PE column
Column Packed Height: 30 cms to 50 cms
-20 Column Volume (CV): lOOmL
Column Max Pressure: IMpa
Solvents Used: Mobile A: Di Water (pH 3.5 to 8)
Mobile B: 20% Iso Propyl Alcohol (pH 3.5 to 8)
Mobile C: 40% Iso Propyl Alcohol (pH 3.5 to 8)
25 Mobile D: 60% Iso Propyl Alcohol (pH 3.5 to 8)
*pH adjusted by 6N HC1.
Sequential Steps:
I. Equilibration: 2CV with Di water (pH 3.5 to 8)
II. Sample Binding: 1.75 CV of Permeate obtained from the membrane fdtration; permeate 30 obtained from microfdtration (pre -fdtration technique); or initial wash flow through obtained from IEX
III. Initial Wash: 1.35 CV with DI water (Initial Wash Flow Through) pH 3.5
IV. Elution: Step Elution i. 2CV with 20% IPA pH 3.5 ii. 2CV with 40% IPA pH 3.5 iii. 2CV with 60% IPA pH 3.5
V. Final Washing: 1 5CV with 80% IPA pH 3.5
Fraction Collection: 12 fractions of 50mL each when the samples were eluted using 20%, 5 40% and 60%IPA. c) Hydrophobic Interaction Chromatography
- Resin Used: Sepabeeds SP700 from Mitsubishi Chemicals, or Sepabeads SP 825L from Mitsubishi Chemicals, or Amberlite XAD 2 from Merck - Supelco, or Amberlite XAD 7HP
10 from Merck - Supelco
Column Used: Glass Column Vantage U- 22 x 600mm, Merck Millipore, any other glass columns, Steel column, Polypropylene (PP) column, or HD PE column
Column Packed Height: 50 cms
Column Volume (CV): lOOmE
-15 Column Max Pressure: IMpa
Solvents Used: Mobile A: Di Water
Mobile B: 100% Ethanol
Sequential Steps:
I. Equilibration: 2CV with DI water
II. 20 Sample Binding: 1.75 CV of permeate obtained from membrane filtration; or permeate obtained from microfdtration (pre -fdtration technique); or initial wash flow through obtained from IEX
III. Initial Wash: 1.75 CV with DI water
IV. Elution: Step Elution
25 2CV with 20% Ethanol ii. 2CV with 40% Ethanol iii. 2CV with 60% Ethanol
V. Final Washing: 1.5CV with Di water
Fraction Collection: 12 fractions of 50mL each when the samples were eluted using 20%, 30 40% and 60% Ethanol.
In some embodiments, the methods for purification of phytochemicals from areca wash liquid (AWL) as described above comprises subjecting the areca wash liquid to a prefiltration step using Whatman filter paper, depth filter, microfiltration, centrifugation, or any combination thereof, before subjecting said areca wash liquid to membrane filtration, chromatography, or the combination of membrane filtration and chromatography.
In some embodiments of the present disclosure, the above described methods for purification of polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL) is carried out at a temperature ranging from about 18°C to about 68°C.
In some embodiments of the present disclosure, the above described methods for purification of polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL) is carried out at a pressure ranging between 1 bar to 20 bars.
In some embodiments of the present disclosure, the above described methods for purification of polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL) is carried out for a time-period ranging from about 0.5 hours to about 20 hours.
In some embodiments of the present disclosure, the above described methods for purification of polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL) is carried out at a temperature ranging from about 18 °C to about 68 °C at a pressure ranging from 1 bar to 20 bars and for a time-period ranging from about 0.5 hours to about 20 hours.
In some embodiments of the present disclosure, the above described methods for purification of phytochemicals - polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL), results in said phytochemicals having a purity ranging from about 65% to about 100%.
In some embodiments of the present disclosure, the above described methods for purification of polyphenol selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof, from areca wash liquid (AWL), results in polyphenol having a purity ranging from about 65% to about 100%. In some embodiments, the above described methods for purification of ursolic acid (triterpene) or its isomers, from areca wash liquid (AWL), results in ursolic acid having a purity ranging from about 65% to about 100%. In some embodiments of the present disclosure, the above described methods for purification of catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL), results in catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, or any combination thereof, having a purity of about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%,
93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%,
99.5%, 99.9%, or 100%.
In some embodiments of the present disclosure, the above described methods for purification of arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL) results in arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, having a purity ranging from about 65% to about 100%. In some embodiments of the present disclosure, the above described methods for purification of arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL) results in arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, having a purity of about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, or 100%.
In some embodiments of the present disclosure, the above described methods for purification of polyphenol(s), alkaloid(s), triterpene(s), or any combination thereof, from areca wash liquid (AWL), results in polyphenols, alkaloid and/or triterpene(s) with an yield ranging from about 1.5% to about 85%.
In some embodiments of the present disclosure, the above described methods for purification of arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL) results in a yield ranging from about 1.5% to about 5.5%. In some embodiments of the present disclosure, the above described methods for purification of catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, or any combination thereof, from areca wash liquid (AWL) results in a yield ranging from about 5% to about 85%. In some embodiments of the present disclosure, the above described methods for purification of ursolic acid from areca wash liquid (AWL) results in ursolic acid yield ranging from about 0.2% to about 5%.
In some embodiments of the present disclosure, the other mentioned compounds described herein, and the isomers of such compounds are also isolated resulting in a yield ranging from about 5% to about 30%.
Thus, the present disclosure successfully employs areca wash liquid (AWL) as the raw material/input for isolating industrially valuable components including polyphenols, alkaloids and/or triterpene(s). Polyphenols such as catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid and resveretrol, and alkaloids such as arecoline, arecaidine, guvacine and guvacoline are important phytochemical molecules present in higher quantity in the AWL than in the arecanuts that are marketed and exported to foreign countries. EGCGs and catechin are polyphenolic antioxidants whereas, arecoline is a naturally derived free basic alkaloid. Applications of EGCG and catechin are majorly in industries such as Nutraceutical, Cosmetics and Skincare, Pharmaceutical, Food and Beverages, and Consumer electronics. These molecules exhibit properties such as antioxidative property, glucosidase inhibitory property and anti-hypertensive property. Arecoline exhibits parasympathetic and muscarinic properties and is a cholinergic agent. Arecoline is majorly used in sports supplements which help individuals to stay active and energetic and in some special dietary supplements that help in improvising the learning ability and neural activity of people with neural/leaming disorders. Arecoline being an anthelminthic drug also finds its application in animal and pet anthelminthic formulations. Hence, keeping in mind the chemical and molecular nature of the above mentioned important natural phytochemicals (polyphenols, alkaloids and triterpenes), the present disclosure provides a method for isolation/purification of the said phytochemicals from an economical raw material - areca wash liquid (AWL). The products obtained from the purification methods described herein is selected from a) an alkaloid product comprising either arecoline, arecaidine, guvacine, guvacoline or any combination thereof; b) a polyphenol product comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), 5 syringic acid, coumaric acid, resveretrol, or any combinations thereof; c) a product comprising a) and b); or d) a product comprising arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid and resveretrol.
10 Further, the method of the present disclosure employs steps including but not limited to membrane processes such as ultrafiltration, microfiltration, depth filtration, filter paper based clarification, or any combination thereof; chromatographic processes such as cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography (using polymeric macroporous hydrophobic resins), other adsorption 15 chromatographic techniques, or any combinations thereof.
The method of the present disclosure possesses advantages including but not limited to:
A. the method employs an economical/cost-effective raw material or input (areca wash liquid) for isolating/purifying industrially important phytochemicals (polyphenols and alkaloids),
20 for instance, arecoline, arecaidine, guvacine, guvacoline, catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid and resveretrol. Said raw material (areca wash liquid) has not been employed before for isolating or purifying polyphenols and/or alkaloids. In fact, the areca wash liquid is considered majorly a waste material and is discarded after agro- 25 processing of arecanuts;
B. AWL can pose a threat to the environment if discarded into soil. Particularly, it can cause acidification of the soil because of the acidic pH of the material (AWL).
C. the present method employs an alternate source/raw material for isolating polyphenols compared to conventional raw materials or sources (eg. green tea, gambier, acacia). Isolation
30 from such conventional raw materials (eg. green tea, gambier, acacia) makes the overall process costly and complex when compared to AWL (waste material); and
D. purification method of the present disclosure employs simple and green/environmentally friendly process conditions/parameters and avoids usage of harsh conditions. It is to be understood that the foregoing descriptive matter/embodiments are illustrative of the disclosure and not a limitation. While considerable emphasis has been placed herein on the particular/preferable features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred 5 embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.
10
Descriptions of well-known/conventional methods/steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples 15 used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.
20 EXAMPLES
Example 1: Preparation and characterization of areca wash liquid (AWL)
Fresh areca wash liquid (AWL) was obtained from various cultivators/farmers in Yellapur, North Canara district of Karnataka. Briefly, said cultivators/farmers prepared the areca wash liquid (AWL) by:
£ 5 harvesting the areca fruits from the areca palm, b) dehusking the areca fruit by separating the husk and the kernel, c) whole nuts were transferred to a vessel containing water (water was 3 times in volume of the nuts), d) the whole nuts were boiled for a time period of about 3 hours to 4 hours and was left till it 30 cooled down, e) the whole nuts were separated from the water by draining them, and the separated whole nuts were sun dried and stored in bags, f) the drained/separated water of step e) was the areca wash liquid (AWL) which can be used for isolation of alkaloids and/or polyphenols. In the present experiment, the obtained AWL was further reused for boiling fresh dehusked arecanuts. g) The AWL was used for boiling 2-3 batches of fresh whole arecanuts with the addition of 5 extra volume of water. h) The obtained AWL from step g) was thereafter stored in a concrete container or HDPE canisters and was used as a raw material for further experiments.
The fresh AWL was transported in PET cans to the R&D premises at Bangalore 10 Bioinnovation Centre (BBC), Bengaluru. The AWL was subjected to Phytochemical analysis (Qualitative analysis) to determine the overall constituents/composition of AWL. Particularly, tests for Alkaloids, Flavonoids, Tannins, Protein and Carbohydrates were performed. Quantitative tests included test for polyphenols, i.e., determination of total polyphenolic content in the AWL by using the standard Folin-Ciocalteu reagent and by 15 measuring the absorbance at 765nm by using gallic acid as standard. From the qualitative analysis of the AWL, the presence of alkaloids, flavonoids, tannins, saponins and minute traces of carbohydrates were detected. The flavonoids consisted of majorly polyphenols belonging to the group of epigallocatechin (EGC), catechin, epicatechin, epigallocatechin gallate (EGCG), syringic acid, epicatechin gallate (EGC) at higher concentrations, and 20 resveratrol, quercetin in minor concentrations.
Example 2: Purification of alkaloids and polyphenols by ion exchange chromatography (IEX), membrane filtration, and hydrophobic interaction chromatography (HIC)
The AWL obtained in Example 1 was used for isolation/purification of the compounds of 25 interest (alkaloids and polyphenols) as follows:
Pre-filtration:
Centrifugation - AWL samples were centrifuged at 24,000 rpm for about 20 minutes. For industrial/commercial scale, continuous centrifugation may be employed to handle sample 30 volumes of a large scale.
The prefiltered AWL was subjected to sequential processes of ion exchange chromatography (IEX), membrane filtration (microfiltration followed by ultrafiltration), and hydrophobic interaction chromatography (HIC) as follows: 1. Ion exchange chromatography (IEX):
Column used: Vantage Glass column L- 16 x 500mm, Merck Millipore
Resins: Diaion SK1B- Mitsubishi chemicals (Cation Exchange Resin)
Column packed: 30 cms
5 Sample: prefiltered AWL (Centrifuged at 24,000 rpm for about 20 min)
Equilibration Buffers: Di Water (pH 3.5 to 4)
Elution Buffers: 2%, 3% and 4% NaCl,
Column Regeneration buffer: 3% HC1
Sequential Steps:
3L0 Column Equilibration: 2CV (Column Volume) b. Sample Loading: 2CV c. Intermediate Washing: DI Water pH (3.5 to 4) 2CV d. Elution: Step Elution using the elution Buffers: 6CV [2% NaCl, 2CV; 3% NaCl, 2CV and 4% NaCl, 2CV],
<15 Final wash or Column regeneration: 3% HC1, 2CV
2. Membrane Filtration:
Micro filtration -
0.45p Hydrosart (cellulose based) membrane (Sartorius)
20 Sample: Initial wash flow through (obtained from IEX) without any dilution with any polar solvent
TMP: 1.5 bar
Total time: about 8 hours (10L of raw material/sample)
25 Ultrafiltration -
IKDa Polyethersulfone (PES) membrane (Sartorius)
Sample: Undiluted permeate from microfiltration
TMP: 1 bar
Total time: about 48 hours
30
3. HIC:
Column used: Vantage Glass column L- 22 x 500mm, Merck Millipore
Resin: Sepabeeds SP700 - Mitsubishi chemicals (Macroporus Resin) Column packed: 30 cms Sample: Permeate obtained from Ultrafiltration
Equilibration Buffers: DI Water (pH 3.5)
Elution Buffers: 20-60% IPA
Column Regeneration: 85% IPA
5 Sequential Steps: a. Column Equilibration: 2CV (Column Volume) b. Sample Loading: 2CV c. Intermediate Washing: DI Water pH (3.5) 2CV d. Elution: Step Elution using the elution Buffers: 6CV, pH 3.5.
10 Step Elution: 20% ethanol/ IPA: 2CV
40% ethanol/IPA: 2CV
60% ethanol/IPA: 2CV e. Final wash or Column regeneration: 2CV
An illustration of this purification method is shown in Figure 1.
15
Example 3: Purification of alkaloids and polyphenols by membrane filtration, hydrophobic interaction chromatography (HIC) and ion exchange chromatography HEX)
The pre-filtered AWL of Example 2 was used for isolation/purification of the compounds of 20 interest (alkaloids and polyphenols). The process conditions/parameters of membrane filtration (MF), hydrophobic interaction chromatography (HIC) and ion exchange chromatography (IEX) remain the same as described in Example 2. The samples used in each process vary which is as follows:
1. MF:
1 5 Microfiltration: Sample: Prefiltered AWL (centrifuged at 24,000 rpm for 20 minutes) and diluted with 10% ethanol b. Ultrafiltration: Undiluted Permeate obtained from microfiltration
2. HIC: a. Sample: Permeate obtained from ultrafiltration. pH adjusted between 3 to 8.
20 IEX: a. Sample: Initial wash flow through (from the intermediate wash) obtained from HIC.
An illustration of this purification method is shown in Figure 2. Example 4: Purification of alkaloids and polyphenols by membrane filtration, ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC) The pre-filtered AWL of Example 2 was used for isolation/purification of the compounds of interest (alkaloids and polyphenols). The process conditions/parameters of membrane 5 filtration (MF), ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC) remain the same as described in Example 2. The samples used in each process vary which is as follows:
1. MF: a. Microfiltration: Sample: prefiltered AWL (centrifuged at 24,000 rpm for 20 minutes) and 10 diluted with 10% ethanol b. Ultrafiltration: Undiluted permeate obtained from microfiltration
2. IEX: a. Sample: Permeate obtained from ultrafiltration. pH adjusted between 3 to 8.
3. HIC: al 5 Sample: Initial wash flow through (from the intermediate wash) obtained from IEX.
An illustration of this purification method is shown in Figure 3.
Example 5: Purification of alkaloids by membrane filtration and ion exchange chromatography (IEX)
20 The pre-filtered AWL obtained in Example 2 was used for isolation/purification of alkaloids. The process conditions/parameters of membrane filtration (MF) and ion exchange chromatography (IEX) remain the same as described in Example 2. The samples used in these processes vary which is as follows:
1. MF:
25 Microfiltration: Sample: prefiltered AWL (centrifuged at 24,000 rpm for 20 minutes) and diluted with 10% ethanol
Ultrafiltration: Undiluted permeate obtained from microfiltration
2. IEX:
Sample: Permeate obtained from ultrafiltration. pH adjusted between 3 to 8.
30 An illustration of this purification method is shown in Figure 4. Example 6: Purification of alkaloids by ion exchange chromatography (IEX)
The pre-filtered AWL obtained in Example 2 was used for isolation/purification of alkaloids. The process conditions/parameters of ion exchange chromatography (IEX) remain the same as described in Example 2. The sample used in this experiment is as follows:
1)5 IEX:
Sample: Prefiltered AWL (Centrifuged at 24,000 rpm for about 20 minutes). This step of centrifugation was carried out twice to get rid of any solid impurities that might clog the resins packed during IEX.
10 Also, the sample may be diluted with Di water or ethanol or IPA if it is too viscous.
An illustration of this purification method is shown in Figure 5.
Example 7: Purification of polyphenols by membrane filtration and hydrophobic interaction chromatography (HIC)
15 The pre-filtered AWL obtained in Example 2 was used for isolation/purification of polyphenols. The process conditions/parameters of membrane filtration (MF) and ion exchange chromatography (IEX) remain the same as described in Example 2. The samples used in these processes were as follows:
1. MF:
120 Microfiltration: Sample: prefiltered AWL (centrifuged at 24,000 rpm for about 20 minutes) and diluted with 10% ethanol c. Ultrafiltration: Undiluted permeate obtained from microfiltration
2. HIC:
Sample: Permeate obtained from ultrafiltration. pH adjusted between 3 to 8.
25 An illustration of this purification method is shown in Figure 6.
Example 8: Purification of polyphenols by hydrophobic interaction chromatography (HIC)
The pre-filtered AWL obtained in Example 2 was used for isolation/purification of 30 polyphenols. The process conditions/parameters of hydrophobic interaction chromatography
(HIC) remain the same as described in Example 2. The sample used in this experiment is as follows:
1) HIC: Sample: Prefiltered AWL (Centrifuged at 24,000 rpm for about 20 minutes). This step was carried out twice to get rid of any solid impurities that might clog the resins packed during HIC.
Also, the sample may be diluted with DI water or ethanol or IPA if it is too viscous.
An illustration of this purification method is shown in Figure 7.
Example 9: HPLC analysis of the purified compounds from AWL
The purified compounds obtained from areca wash liquid (AWL) by employing methods described in Examples 2-8 were analysed/confirmed by High Performance Liquid Chromatography (HPLC).
The HPLC method was optimized on a Waters Automated HPLC, with quaternary pump, auto sampler, PDA detector using a reverse phase C-18 Column. The following conditions were employed:
Mobile phase contained: Acetonitrile (ACN), Isoproyl alcohol(IPA), orthophosphoric acid, Di Water and Methanol [Mobile phase A: 0.1% orthophosphoric acid; Mobile phase B: ACN:IPA (95:5); Di Water and Methanol was used for final column washing]. Flow rate: 1 ml/min
Standard. Sample size and preparation: Standard polyphenols (0.25-lmg/mL) such as catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), Epigallocatechin gallate(EGCG), Quercetin, Syringic acid, and Alkaloid: arecoline was run.
Detector: Wavelength of 216n m for alkaloids and 280 nm for polyphenols was used. Gradient Method:
Table A
Figure imgf000049_0001
Table B
Figure imgf000050_0001
Sample size was optimized by varying volume of injection and sample dilution. Samples were diluted from lx to lOx and volumes were taken from lOpl to lOOpl to get the optimal peak shapes, resolution and quantification.
The chromatograms provided in Figures 8A and 8B show the presence of alkaloid and polyphenols in the AWL. Further, the AWL was subjected to purification by membrane filtration and the final separation of alkaloids (arecoline) and polyphenols such as catechin, epicatechin and epigallocatechin (EGC) were carried out through IEX and HIC respectively.
The chromatograms indicating purification by membrane filtration, IEX and HIC are depicted in Figures 9 to 14 wherein description of each of these chromatograms is provided under ‘BRIEF DESCRIPTION OF FIGURES’. Other polyphenols such as EGCG, ECG, syringic acid, coumaric acid, resveratrol, ursolic acid and alkaloids such as arecaidine, guvacine and guvacoline were present in minute quantities and can be seen with small peaks that are present in these chromatograms (Figures 9 to 14).
As regards all the embodiments characterized in this specification, it is intended that each embodiment mentioned herein can be combined with other embodiments. As an example, in case of an embodiment reciting 3 alternatives A, B and C, and a subsequent or a previous embodiment reciting alternatives D, E and F, it is to be understood that the specification unambiguously discloses embodiments corresponding to all possible combinations, such as, A, B, C; A, B, D; A, B, E; A, B, F; A, C, D; A, C, E; A, C, F; A, B, C, D, E, F etc., unless specifically mentioned otherwise. The above considerations apply mutatis mutandis to all embodiments/features of the present specification. The foregoing description of the specific embodiments reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “essentially comprises”, “including” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Additionally, throughout the specification, “a group comprising” of a particular stated element, integer or step, or group of elements, integers or steps also envisages “a group consisting” of said stated element, integer or step, or group of elements, integers or steps without inclusion of any other element, integer or step, or group of elements, integers or steps. Throughout this specification, the term “about” encompasses minor variations of the specified value. These minor variations would be apparent to one of ordinary skill in the art in the context of the invention. With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.
INCORPORATION BY REFERENCE
Any discussion on references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

We Claim:
1. A method for purification of phytochemicals from areca wash liquid (AWL), said method comprising subjecting the areca wash liquid (AWL) to membrane filtration, or chromatography, or a combination of membrane filtration and chromatography; wherein the phytochemical is at least one of alkaloid, polyphenol and triterpene.
2. The method as claimed in claim 1, wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof; the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof; and the triterpene is ursolic acid, isomers of ursolic acid, or a combination thereof.
3. The method as claimed in claim 1, wherein the areca wash liquid (AWL) is obtained by a process comprising: a) de-husking areca fruits to obtain arecanuts; b) boiling the arecanuts (whole nuts) in presence of a solvent and optionally a base; and c) separating liquid from the arecanuts after step b), wherein said liquid is the areca wash liquid (AWL); or, a) de-husking areca fruits to obtain arecanuts; b) boiling the arecanuts (whole nuts) in presence of a solvent and optionally a base; and c) separating liquid from the arecanuts after step b), d) boiling fresh de-husked arecanuts (whole nuts) in presence of the solvent, the separated liquid of step c) and optionally the base, e) separating liquid from the arecanuts after step d), wherein said liquid is the areca wash liquid (AWL), and wherein said step d) and step e) are optionally repeated for about 1 to 5 times. The method as claimed in claim 3, wherein step b) or step d) is earned out at a temperature of about 50°C to 100°C for a time-period ranging from about 1 hour to 12 hours; wherein the solvent in the process is water, a polar solvent, or a mixture thereof; wherein the polar solvent is selected from a group comprising ethanol, methanol, ethyl acetate, isopropyl alcohol and mixtures thereof; and wherein the base in the process is calcium hydroxide. The method as claimed in claim 3 or claim 4, wherein the areca wash liquid (AWL) is obtained by a process comprising: a) de-husking areca fruits to obtain arecanuts; b) boiling the whole arecanuts in water at a temperature of about 65 °C to about 100°C for a time-period of about 2 hours to 5 hours; and c) separating liquid from the arecanuts after step b), wherein said liquid is the areca wash liquid (AWL); or, a) de-husking areca fruits to obtain arecanuts; b) boiling the whole arecanuts in water at a temperature of about 65 °C to about 100°C for a time-period of about 2 hours to 5 hours; and c) separating liquid from the arecanuts after step b), d) boiling fresh de-husked arecanuts (whole nuts) in fresh water and the separated liquid of step c) at a temperature of about 65°C to about 100°C for a time-period of about 2 hours to 5 hours, e) separating liquid from the arecanuts after step d), wherein said liquid is the areca wash liquid (AWL), and wherein said step d) and step e) are optionally repeated for about 1 to 5. The method as claimed in claim 1, wherein the areca wash liquid (AWL) essentially comprises: a) polyphenols, b) alkaloids, c) saponins, d) triterpenes and steroids, e) polysaccharides, f) minerals, and g) optionally, a residual component selected from a group comprising sugars, proteins, tannin aggregates, fat, impurities and combinations thereof.
7. The method as claimed in claim 6, wherein the areca wash liquid (AWL) essentially comprises polyphenols at a concentration ranging from about 15 wt % to 70 wt %, alkaloids at a concentration ranging from about 0.5 wt % to 4.5 wt %, polysaccharides at a concentration ranging from about 12 wt % to about 30 wt %, minerals at a concentration ranging from about 0.2 wt % to about 2 wt %, and residual component at a concentration ranging from about 0.3 wt % to about 1.5 wt %.
8. The method as claimed in claim 1, wherein the membrane filtration comprises cross flow filtration (CFF), depth filtration, or a combination thereof, and wherein the cross flow filtration (CFF) is selected from ultrafiltration (UF), microfiltration (MF), and a combination of ultrafiltration (UF) and microfiltration (MF); the chromatography is selected from ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC); and wherein the ion exchange chromatography (IEX) is cation exchange chromatography, anion exchange chromatography, or both.
9. The method as claimed in any of the preceding claims, wherein the ion exchange chromatography (IEX) purifies alkaloid; the hydrophobic interaction chromatography (HIC) purifies polyphenol; and the membrane filtration separates large molecules comprising at least one of sugars, proteins, tannin aggregates, fats, impurities, and combinations thereof.
10. The method as claimed in claim 1, wherein the areca wash liquid (AWL) is subjected to prefiltration before subjecting the areca wash liquid to membrane filtration, or chromatography, or the combination of membrane filtration and chromatography; and wherein said prefiltration comprises centrifugation, microfiltration, ultrafiltration, filtration using Whatman filter paper, filtration using depth filter, or any combination thereof.
11. The method as claimed in any of the preceding claims, wherein the method purifies alkaloids and polyphenols comprising steps of: i) subjecting the areca wash liquid to ion exchange chromatography (IEX) to obtain initial wash flow through liquid (IWFT) and eluate, wherein the eluate comprises the alkaloid, and 11) subjecting the initial wash flow through liquid (IWFT) to membrane filtration, followed by hydrophobic interaction chromatography (HIC) to obtain eluate comprising the polyphenols; or i) subjecting the areca wash liquid to membrane filtration followed by ion exchange chromatography (IEX) to obtain initial wash flow through liquid (IWFT) and eluate, wherein the eluate comprises the alkaloid, and ii) subjecting the initial wash flow through liquid (IWFT) to hydrophobic interaction chromatography (HIC) to obtain eluate comprising the polyphenol; or i) subjecting the areca wash liquid to membrane filtration followed by hydrophobic interaction chromatography (HIC) to obtain initial wash flow through liquid (IWFT) and eluate, wherein the eluate comprises the polyphenol, and ii) subjecting the initial wash flow through liquid (IWFT) to ion exchange chromatography (IEX) to obtain an eluate comprising the alkaloid; wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof; and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof. The method as claimed in any of the preceding claims, wherein the method purifies alkaloids and polyphenols comprising steps of: i) subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the alkaloids, wherein the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, b) loading areca wash liquid (sample) onto the column and binding at a pH between 3.5 to 10.5, c) intermediate wash of the column with the equilibration buffer of step a) to obtain initial wash flow through liquid (IWFT), and d) elution of the column using an elution buffer of pH between 2 to 11 to obtain an eluate comprising the alkaloid; and ii) subjecting the initial wash flow through liquid (IWFT) obtained from the intermediate wash of the ion exchange chromatography (step c) to membrane filtration, followed by hydrophobic interaction chromatography (HIC) to purify the polyphenols, wherein the membrane filtration comprises steps of: a) equilibrating a polymeric membrane with solvent, b) applying the initial wash flow through liquid (sample) onto the polymeric membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, and c) washing and re-equilibrating the polymeric membrane, and wherein the hydrophobic interaction chromatography (HIC) comprises steps of: a) equilibrating a HIC column with an equilibration buffer having a pH of 3.5 to 10.5, b) loading the permeate (sample) onto the HIC column, and intermediate wash of the column using the equilibration buffer of step a), and c) elution of the column using an elution buffer of pH of 2.5 to 10.5 to obtain an eluate comprising polyphenol; or i) subjecting the areca wash liquid to membrane fdtration followed by ion exchange chromatography (IEX) to purify the alkaloids, wherein the membrane fdtration comprises steps of: a) equilibrating a polymeric membrane with solvent, b) applying the areca wash liquid (sample) onto the membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, c) washing and re-equilibrating the membrane, and the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, b) loading the permeate (sample) onto the column and binding at a pH between 3.5 to 10.5, c) intermediate wash of the column with the equilibration buffer of step a) to obtain initial wash flow through liquid (IWFT), d) elution of the column using an elution buffer of pH between 2 to 11 to obtain eluate comprising the alkaloid; and ii) subjecting the initial wash flow through liquid (IWFT) obtained from the intermediate wash of the ion exchange chromatography (step c) to hydrophobic interaction chromatography (HIC) to purify the polyphenols, wherein the hydrophobic interaction chromatography (HIC) comprises steps of: a) equilibrating a HIC column with an equilibration buffer having a pH of 3.5 to 10.5, b) application of the initial wash flow through liquid (sample) onto the HIC column, and intermediate wash using the equilibration buffer of step a), and c) elution of the HIC column using an elution buffer of pH of 2.5 to 10.5 to obtain an eluate comprising the polyphenol; or i) subjecting the areca wash liquid to membrane filtration followed by hydrophobic interaction chromatography (HIC) to purify the polyphenols, wherein the membrane filtration comprises steps of: a) equilibrating a polymeric membrane with solvent, b) applying the areca wash liquid (sample) onto the polymeric membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, c) washing and re-equilibrating the polymeric membrane, and wherein the hydrophobic interaction chromatography (HIC) comprises steps of: a) equilibrating a HIC column with an equilibration buffer having a pH of 3.5 to 10.5, b) application of the permeate (sample) onto the HIC column, and intermediate wash using the equilibration buffer of step a) to obtain initial wash flow through liquid (IWFT), and c) elution of the HIC column using an elution buffer of pH of 2.5 to 10.5 and obtaining eluate comprising the polyphenol, and ii) subjecting initial wash flow through liquid (IWFT) obtained from the intermediate wash of hydrophobic interaction chromatography (HIC) (step b) to purify the alkaloids, wherein the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, b) loading the initial wash flow through liquid (sample) onto the column, and binding at a pH between 3.5 to 10.5, c) intermediate wash of the column with the equilibration buffer of step a), and d) elution of the column using an elution buffer of pH between 2 to 11 and obtaining eluate comprising the alkaloid; wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof; and the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
13. The method as claimed in any of the preceding claims, wherein the method purifies alkaloids comprising steps of: i) subjecting the areca wash liquid to membrane filtration and obtaining a permeate, and ii) subjecting the permeate to ion exchange chromatography (IEX) to obtain an eluate comprising the alkaloids; or, subjecting the areca wash liquid to ion exchange chromatography (IEX) to obtain an eluate comprising the alkaloids; wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof.
14. The method as claimed in any of the preceding claims, wherein the method purifies alkaloids comprising steps of: i) subjecting the areca wash liquid to membrane filtration and obtaining a permeate, wherein the membrane filtration comprises steps of: a) equilibrating a polymeric membrane with solvent, b) applying the areca wash liquid (sample) onto the polymeric membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, and c) washing and re-equilibrating the polymeric membrane, and ii) subjecting the permeate to ion exchange chromatography (IEX) to purify the alkaloids, wherein the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, b) loading the permeate (sample) onto the column and binding at a pH between 3.5 to 10.5, c) intermediate wash of the column with the equilibration buffer of step a) to obtain initial wash flow through liquid (IWFT), and d) elution of the column using an elution buffer of pH between 2 to 11 and obtaining eluate comprising the alkaloid; or, subjecting the areca wash liquid to ion exchange chromatography (IEX) to purify the alkaloids, wherein the ion exchange chromatography comprises steps of: a) equilibrating a cation exchange column or an anion exchange column with an equilibration buffer of pH between 3.5 to 10.5, wherein the cation exchange column is packed with a cation exchange resin and the anion exchange column is packed with an anion exchange resin, b) loading the areca wash liquid (sample) onto the column and binding at a pH between 3.5 to 10.5, c) intermediate wash of the column with the equilibration buffer of step a), and d) elution of the column using an elution buffer of pH between 2 to 11 and obtaining an eluate comprising the alkaloid; wherein the alkaloid is selected from a group comprising arecoline, arecaidine, guvacine, guvacoline, isomers of said compounds, and combinations thereof. The method as claimed in any of the preceding claims, wherein the method purifies polyphenols comprising steps of: i) subjecting the areca wash liquid to membrane filtration and obtaining a permeate, and ii) subjecting the permeate to hydrophobic interaction chromatography (HIC) to obtain an eluate comprising the polyphenol; or, subjecting the areca wash liquid to hydrophobic interaction chromatography (HIC) to obtain an eluate comprising the polyphenols; wherein the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof.
16. The method as claimed in any of the preceding claims, wherein the method purifies polyphenols comprising steps of: i) subjecting the areca wash liquid to membrane filtration and obtaining a permeate, wherein the membrane filtration comprises steps of: a) equilibrating a polymeric membrane with solvent, b) applying the areca wash liquid (sample) onto the polymeric membrane to obtain a permeate comprising compounds of interest and a retentate comprising impurities, and c) washing and re-equilibrating the polymeric membrane, and ii) subjecting the permeate to hydrophobic interaction chromatography (HIC) to purify the polyphenols, wherein the hydrophobic interaction chromatography (HIC) comprises steps of: a) equilibrating a HIC column with an equilibration buffer having a pH of 3.5 to 10.5, b) loading the permeate (sample) onto the column, and intermediate wash of the column using the equilibration buffer of step a), and c) elution of the column using an elution buffer of pH of 2.5 to 10.5 to obtain an eluate comprising the polyphenol; or, subjecting the areca wash liquid to hydrophobic interaction chromatography (HIC) to purify the polyphenols, wherein the hydrophobic interaction chromatography (HIC) comprises steps of: a) equilibrating a HIC column with an equilibration buffer having a pH of 3.5 to 10.5, b) loading the areca wash liquid (sample) onto the column, and intermediate wash of the column using the equilibration buffer of step a), and c) elution of the column using an elution buffer of pH of 2.5 to 10.5, to obtain an eluate comprising the polyphenol; wherein the polyphenol is selected from a group comprising catechin, epicatechin, epigallocatechin (EGC), Epicatechin Gallate (ECG), epigallocatechin gallate (EGCG), syringic acid, coumaric acid, resveretrol, isomers of said compounds, and combinations thereof. The method as claimed in any of the preceding claims, wherein said method for purification of phytochemicals from areca wash liquid (AWL) is carried out at a temperature ranging from about 18°C to 68°C at a pressure ranging from 1 bar to 20 bars and for a time-period ranging from about 0.5 hours to 100 hours, wherein the phytochemical is at least one of alkaloid, polyphenol and triterpene. The method as claimed in any of the preceding claims, wherein said method for purification of phytochemicals from areca wash liquid (AWL) results in phytochemicals having a purity ranging from about 65% to 100%, and a yield ranging from about 1.5% to 85%, wherein the phytochemical is at least one of alkaloid, polyphenol and triterpene.
60
PCT/IB2022/050287 2021-01-14 2022-01-14 Purification of phytochemical(s) from areca wash liquid WO2022153228A1 (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
US5886155A (en) * 1997-06-18 1999-03-23 Bioresources International Inc. Purification of miraculin glycoprotein using tandem hydrophobic interaction chromatography
CN102464607A (en) * 2010-11-19 2012-05-23 苏州宝泽堂医药科技有限公司 Method for extracting arecoline from areca

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US5886155A (en) * 1997-06-18 1999-03-23 Bioresources International Inc. Purification of miraculin glycoprotein using tandem hydrophobic interaction chromatography
CN102464607A (en) * 2010-11-19 2012-05-23 苏州宝泽堂医药科技有限公司 Method for extracting arecoline from areca

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Title
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