Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C227/38—Separation; Purification; Stabilisation; Use of additives
  • C07C227/40—Separation; Purification
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/02—Nutrients, e.g. vitamins, minerals

Definitions

• the present invention relates to an improved process for purification of free bio amino acids (theanine enriched) from parts of plants, preferably from tea (Camellia sinensis).
• the invention relates to an economical process for purification of natural free amino acids from plant and plant parts like leaves, flowers and fruits (renewable bioresources) without using any chemical substance, acid or alkali.
• the invention relates to a low cost, green process for purification of fraction containing natural free amino acids; to be used as readily bioavailable nutritional supplements, especially for vegetarians and protein deficient people as an option for costlier protein supplements.
• the amino acids are the building blocks of proteins and act as intermediates in body metabolism.
• the importance of amino acids as building blocks of proteins for the growth and repair of muscles, bones, skin, tendons, ligaments, hair, eyes and other tissues is proven since a very long time.
• Proteins play a vital role in various body functions such as, metabolism, cell signalling, the immune system, cell adhesion and cell cycle.
• the problem of protein deficiency is very common among populations of poor, undeveloped and in developing countries. Even in developed countries, this problem of protein deficiency was reported in certain groups of population with specific diet habits. Particularly, vegetarians need to be very careful about eating the right combinations of plant foods to get enough of the essential amino acids.
• proteins enter the body first they are broken down into amino acids during the digestion process.
• amino acids are then used in different areas of the body where they are needed for protein synthesis.
• the amino acids are also responsible for the biological activities of proteins. So, the amino acids which are the building blocks of proteins are vital for body functioning. So there exist huge potential of using amino acids in form of nutritional supplements.
• Tea ⁇ Camellia sinensis of Theaceae family is known world wide as a beverage crop. This plant is also a good source of free amino acids. The amino acids content in the tea was estimated from 3-4% on dry wt. basis. The presence of free amino acids in tea is well known and it was reported that it contains all the 20 amino acids.
• Tea contains all the essential amino acids along with non-essential ones. Along with that, tea also contains a unique non-protein derived amino acid called as theanine which was formed from glutamine and ethylamine (Vuong et al., Journal of Science of Food and Agriculture, 201 1, 91 , 1931-1939).
• Theanine is well known as a psychoactive amino acid which crosses the blood brain barrier and relaxes during stress. High water solubility of theanine in comparison to caffeine and catechins and its presence in sufficient amount in tea plant is an important requisite of the present invention; as it constitutes up to 50% of total amino acids present in the tea.
• Ekanayake et al. (US Patent app. No. 10/689910) has explained a process for isolation of theanine by extraction, absorption and filtration from plant material using isopropanol as a solvent to get theanine enriched extract. They used polyamides, polyvinylpyrollidine and polycar as adsorbants.
• Tea contains all the essential amino acids like, threonine, valine, leucine, isoleucine, lysine, phenylalanine and trytophan in it. So the present invention was specially focused on obtaining all these free amino acids present in tea along with theanine by an economical and green process methodology.
• nutraceutiacls and additives their market is also growing at very fast rate world wide. This might be due to health safety and therapeutic potential associated with them. So, in an attempt to obtain natural bio amino acids by a low cost green methodology and as an option for costlier protein supplements; the present invention was carried out.
• the main objective of the present invention is to develop a green and economical aqueous process for purification of free bio amino acids from plant sources/renewable bioresources.
• Another objective of the present invention is to purify free amino acids of tea along with theanine.
• Figure 1 illustrates a schematic flow chart of process for purification of bio amino acids.
• Figure 3a UPLC Chromatogram of mixture of amino acids standards, Figure 3b; UPLC Chromatogram of tea bio amino acids (theanine enriched) and Figure 3c; UPLC Chromatogram of Sesbania grandiflora bio amino acids.
• the present invention relates to an improved process for purification of free bio amino acids from natural sources like plants and plant parts by a solvent free and economical process.
• the present invention provides an improved process for production of free bio amino acids said process comprising:
• step (b) filtering the aqueous plant extract obtained in step (a) through cotton filters and obtaining filtered aqueous extract
• step (b) passing the filtered aqueous extract of step (b) through a first column packed with polymethacrylate based resin with particle size of 100-300 microns and saturated with water preferably with double distilled water, to get polyphenol free effluent,
• step (c) passing the polyphenol free effluent of step (c) through a second column packed with polystyrene divinylbenzene polymer based resin with particle size of 120 microns, saturated with double distilled water and obtaining the effluent,
• step (d) concentrating the effluent of step (d) containing free bio amino acids.
• bio amino acids ' ' ' ' used herein relates to the purely natural free amino acids fraction (theanine rich) from the tea shoots in particular and from other plant renewable parts like fruits, leaves, flowers, and seeds. These bio amino acids are pure and 100% natural fraction, obtained especially from natural sources like plants and plant parts without using any extraneous chemical substance or acid and alkali.
• tea shoots is used herein for fresh tea leaves along with stem portion.
• completely free from polyphenols means that purified bio amino acids fraction contains no caffeine, lipids, saponins, alkaloids and catechins as detected by RP- HPLC analysis.
• regenerable resources is used herein for leaves, fruits, flowers and seeds of plants and more preferably for leaves.
• w ter refers to distill water, deionized water and more preferably double distilled water.
• the first step of the present invention relates to the aqueous extraction of the fresh tea shoots, which is done by any of the following methods:
• the aqueous extract obtained from different extraction steps is then filtered twice using cotton (adsorbent) filter.
• the purification of free amino acids from the filtered aqueous extract is done by using adsorption column chromatography using two different adsorption resins both of which differs in their chemistry and particle size.
• the adsorption is a surface phenomenon which involves weak and reversible binding of selective compounds or molecules present in the mixture with the surface of stationary phase. So, herein the present invention the free amino acids are purified by selecting two different resins in series and by removing other major components present in the extracts.
• the resin (A) used herein in the present invention is a polymethacrylate based resin preferably sepabeads with particle size of 100-300 microns, while resin (B) used herein in the present invention is a polystyrene divinylbenzene polymer based resin preferably amberchrom with particle size of 120 microns.
• the filtered aqueous extracts were then passed through prepacked resin column (A) polymethacrylate based resin with particle size of 100-300 microns.
• the column was packed and saturated in water more preferably with double distilled water. This resin column adsorbs the polyphenols and other complex molecules allowing only amino acids, proteins and caffeine to pass through along with water as effluent.
• the effluent aqueous fraction contains natural bio amino acids and is completely free from catechins and caffeine which are other major constituents of tea.
• This aqueous fraction containing free bio amino acids theanine enriched in case of tea ( Figure 2a) compared with standard theanine ( Figure 2b), is then concentrated with rotary evaporator followed by spray drying or through lyophilization by freeze drying to obtain off white to light brown colored powder.
• the bio amino acids fraction is further analyzed by paper chromatography and RP-HPLC ( Figure 4a, 4b) and UPLC ( Figure 3b, 3c) analysis.
• the results of RP-HPLC significantly shows the presence of theanine as major component among the tea bio amino acids as major constituents.
• the invention provides a process for purification of free bio amino acids form natural sources like plants and plant parts, especially from those which are rich source of free amino acids.
• the present invention discloses a process developed by using combination of adsorption resins of different particle size and chemistry to purify bio amino acids (theanine enriched) ( Figure 2a) fraction from tea shoots and completely free from polyphenols catechins, lipids, saponins, alkaloids and caffeine.
• the solvent free process for purification of free bio amino acids comprises the following steps:
• step (b) filtering the aqueous plant extract obtained in step (a) through cotton filters and obtaining filtered aqueous extract
• step (b) passing the filtered aqueous extract of step (b) through a first column packed with polymethacrylate based resin with particle size of 100-300 microns and saturated with water preferably with double distilled water, to get polyphenol free effluent,
• step (c) passing the polyphenol free effluent of step (c) through a second column packed with polystyrene divinylbenzene polymer based resin with particle size of 120 microns, saturated with double distilled water and obtaining the effluent,
• step (d) concentrating the effluent of step (d) containing free bio amino.
• the plant parts used is theanine rich leaves or shoots such as tea leaves or shoots or any other plant part which contains free amino acids.
• the extraction is done by any of the following methods: a. Freeze drying the plant parts at -20 ° C, crushing the freezed plant parts to powder form and extracting the freezed and crushed plant parts with warm water (60 ° C).
• polymethacrylate based resin is preferably sepabeads.
• polystyrene divinylbenzene polymer based resin is preferably amberchrom.
• the concentration is done with rotary evaporator followed by spray drying, optionally through lyophillization by freeze drying.
• bio amino acids obtained are completely free from tea polyphenols (catechins), caffeine and other major constituents of tea leaves.
• the present invention described a green process without using chemical substances, acids and alkali to purify free bio amino acids as readily bioavailable source, compared to costly protein supplements.
• This column adsorbs the caffeine, proteins and other impurities allowing bio amino acids to pass through the column along with water.
• this aqueous fraction contains purified bio amino acids. Dry the aqueous fraction to get pure bio amino acids by spray drying.
• the RP-HPLC chromatogram shows the presence of amino acids (theanine enriched) ( Figure 2a) compared with the standard theanine ( Figure 2b).
• bio amino acids are completely free from tea polyphenols (catechins) and caffeine which are other major constituents of tea.
• Purified natural free amino acids can be used as a source of more readily bioavialable health supplements as compared to costly protein supplements.

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• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
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• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Diabetes (AREA)
• Pharmacology & Pharmacy (AREA)
• Life Sciences & Earth Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Medicines Containing Plant Substances (AREA)
• Coloring Foods And Improving Nutritive Qualities (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

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• 2013
  • 2013-02-08 CN CN201380008598.2A patent/CN104105685B/zh not_active Expired - Fee Related
  • 2013-02-08 IN IN371DE2012 patent/IN2012DE00371A/en unknown
  • 2013-02-08 JP JP2014556200A patent/JP6186379B2/ja not_active Expired - Fee Related
  • 2013-02-08 WO PCT/IN2013/000088 patent/WO2013118152A1/en not_active Ceased
  • 2013-02-08 US US14/376,631 patent/US9266818B2/en not_active Expired - Fee Related

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