Method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi

Abstract

The present invention relates to the technical field of Camellia nitidissima Chi processing and application, and provides a method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi. The method comprises the following steps: taking a graphene nano material as a selective extraction, adsorption and separation carrier material; carrying out stepwise separation through stepwise controlling the pH value of Camellia nitidissima Chi concentrated solution and adjusting the isoelectric points of the amino acid active ingredients, wherein the amino acid active ingredients comprise aspartic acid, threonine, serine, glutamic acid, proline and glycine, and the pH values of the aspartic acid, the threonine, the serine, the glutamic acid, the proline and the glycine corresponding to the stepwise separated isoelectric points are less than 2.77, 5.98-6.15, 3.23-5.67, 2.78-3.21, 6.17-6.29 and 5.69-5.96. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi has the characteristics of superior selectivity, superior separation speed, good product purity and low cost.

Claims

1. A method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi, characterized in that it comprises the following steps: utilizing a graphene nano material as a selective extraction, adsorption and separation carrier material; carrying out stepwise separation through stepwise controlling the pH value of a Camellia nitidissima Chi concentrated solution; and adjusting the isoelectric points of the amino acid active ingredients; wherein the amino acid active ingredients comprise aspartic acid, threonine, serine, glutamic acid, proline and glycine.

2. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi according to claim 1, characterized in that the pH values of the aspartic acid, the threonine, the serine, the glutamic acid, the proline and the glycine corresponding to the stepwise separated isoelectric points are less than 2.77, 5.96-6, 15, 3.23-5.67, 2.78-3.21, 6.17-6.29 and 5.69-5.96, respectively.

3. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi according to claim 1, characterized in that the graphene nano materials are selected from graphene oxide, hydroxyl graphene, carboxyl graphene, thiol graphene, graphene modified by chitosan, graphene modified by metal ions, and graphene modified by polymer or biomacromolecules.

4. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi according to claim 3, characterized in that the graphene nano materials are synthesized by renewable resources derived active carbons with an improved Hummers method or reversible addition-fragmentation chain transfer polymerization method.

5. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi according to claim 4, characterized in that the graphene nano materials are cleaned, dried, activated to be recycled after the adsorption of corresponding amino acid steps.

6. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi according to claim 5, characterized in that raw materials to prepare active carbon by carbonizing renewable resources are selected from straw, bagasse and cornstalk.

7. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi according to claim 6, characterized in that the processes of stepwise separation also include increasing separation speed by ultrasonic vibration and dispersion.

8. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi according to claim 2, characterized in that the graphene nano materials are selected from graphene oxide, hydroxyl graphene, carboxyl graphene, thiol graphene, graphene modified by chitosan, graphene modified by metal ions, and graphene modified by polymer or biomacromolecules.

9. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi according to claim 8, characterized in that the graphene nano materials are synthesized by renewable resources derived active carbons with an improved Hummers method or reversible addition-fragmentation chain transfer polymerization method.

10. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi according to claim 9, characterized in that the graphene nano materials are cleaned, dried, activated to be recycled after the adsorption of corresponding amino acid steps.

11. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi according to claim 10, characterized in that raw materials to prepare active carbon by carbonizing renewable resources are selected from straw, bagasse and cornstalk.

12. The method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi according to claim 11, characterized in that the processes of stepwise separation also include increasing separation speed by ultrasonic vibration and dispersion.

Description

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(1) For better understanding of the present invention, the following is detailed description about contents and embodiments of the present invention.

(2) A method for stepwise separating amino acid active ingredients of Camellia nitidissima Chi, it comprises the following steps, taking a graphene nano material as a selective extraction, adsorption and separation carrier material; carrying out stepwise separation through stepwise controlling the pH value Camellia nitidissima Chi concentrated solution and adjusting the isoelectric points of the amino acid active ingredients, wherein the amino acid active ingredients comprise aspartic acid, threonine, serine, glutamic acid, proline and glycine. The method comprise following steps:

(3) Step 1: preparation of Camellia nitidissima Chi concentrated solution: fresh flowers of Camellia nitidissima Chi in autumn were picked and weighted with electronic scale; the picked flowers were chose for the freshest and excellent quality flowers and those with poor quality are removed, the optimized flowers must comply with requirements of Pharmacopoeia of China (2010), that is, no mildew, no odor and no impurity, the optimized flowers of Camellia nitidissima were smashed by grinder after cleaning; some acetone or ethanol with over 95% concentration was added into smashed leaves of Camellia nitidissima Chi and was extracted for 5-6 h by Soxhlet extractor to obtain the first concentrated solution; then some acetone or ethanol with over 95% concentration was added into extracted residue and ultrasound was performed for 1.5 h under 40-60° C. to obtain the second concentrated solution; the first and second concentrated solution was mixed, and acetone or ethanol solvent was removed by rotary evaporation of rotary evaporators, finally the Camellia nitidissima Chi concentrated solution A1 in organic phase was obtained.

(4) Step 2: stepwise separation of proline, comprising following steps:

(5) At first, carrying out pH adjustment, the pH value of Camellia nitidissima Chi concentrated solution obtained in step 1 was adjusted to 6.17-6.29, by this time, the pH value of Camellia nitidissima Chi concentrated solution was less than isoelectric point of the proline only, but more than any other isoelectric points of amino acids, and because when pH>pl, the amino acid was negatively charged, when pH<pl, the amino acid was positively charged, by this time, the proline was positively charged, while the aspartic acid, the threonine, the serine, the glutamic acid, and the glycine were all negatively charged.

(6) Next, carrying out proline selective extraction, some graphene nano materials were added into the Camellia nitidissima Chi concentrated solution A1 after pH adjustment and ultrasound was performed for half an hour, the positively charged proline was combined with the negatively charged graphene nano material assisted by ultrasonic wave, and graphene oxide was taken as the graphene nano material, other negatively charged amino acids were not combined with the graphene nano material, then the Camellia nitidissima Chi concentrated solution B1 was obtained.

(7) Then carrying out proline stepwise separation, the suction filtration is carried out to the Camellia nitidissima Chi concentrated solution B1 after ultrasound to obtain the first filtrate C1 and the first filter residue D1, and the first filter residue D1 was washed with acetone for several times to remove some simple adsorbed non-proline ingredients, while the first filtrate C1 was left to be used for next step, the collected first filter residue D1 was placed into the beaker with 95% ethanol, and ultrasound was performed for an hour, then the second filtrate E1 and the second filter residue F1 were obtained through filtration, the second filter residue F1 was washed with ethanol for several times, after drying and activating, it was left to be subsequent recycled, the ethanol of the second filtrate E1 was removed by rotary evaporation, after drying, the proline product with white crystalline powder can be obtained, besides, the proline product can be further purified through recystallization.

(8) Step 3: stepwise separation of threonine, comprising following steps:

(9) At first, carrying out pH adjustment, the pH value of the first filtrate C1 obtained in step 2 was adjusted to 5.98-6.15 according to the pH adjustment method of step 2, by this time, the threonine was positively charged, while other amino acids were negatively charged;

(10) Next, carrying out threonine selective extraction, some graphene nano materials were added into the first filtrate C1 and ultrasound was performed for half an hour, the positively charged threonine was combined with the negatively charged graphene nano material assisted by ultrasonic wave, and hydroxyl graphene was taken as the graphene nano material, other negatively charged amino acids were not combined with the graphene nano material, then the Camellia nitidissima Chi concentrated solution B2 was obtained.

(11) Then carrying out threonine stepwise separation, the suction filtration was carried out to the Camellia nitidissima Chi concentrated solution B2 to obtain the first filtrate C2 and the first filter residue D2, and the first filter residue D2 was washed with acetone for several times to remove some simple adsorbed non-threonine ingredients, while the first filtrate C2 was left to be used for the second extraction, the collected first filter residue D2 was placed into the beaker with hot water, and ultrasound was performed for half an hour, then the second filtrate E2 and the second filter residue F2 were obtained through immediate filtration, the second filter residue F2 was washed with hot water for several times, after drying and activating, it was left to be subsequent recycled, the second filtrate E2 was placed into the beaker and heated to high temperature, in a successive step, the beaker with the second filtrate E2 was placed into ice water mixture immediately, the threonine product can be obtained after the precipitated white crystal was separated and dried, besides, the product can be further purified through recystallization.

(12) Step 4: stepwise separation of glycine, comprising following steps:

(13) At first, carrying out pH adjustment, the pH value of the first filtrate C2 obtained in step 3 was adjusted to 5.69-5.96 by repeating the pH adjustment, by this time, the glycine was positively charged, while other amino acids were negatively charged;

(14) Next, carrying out glycine selective extraction, some graphene nano materials were added into the second filtrate C2 and the mixed materials of hydroxyl graphene and thiol graphene were taken as the graphene nano material, ultrasound was performed for half an hour, and the positively charged glycine was combined with the negatively charged graphene nano material assisted by ultrasonic wave, then the Camellia nitidissima Chi concentrated solution B3 to be separated was formed.

(15) Then carrying out glycine stepwise separation, the suction filtration was carried out to the Camellia nitidissima Chi concentrated solution B3 to obtain the first filtrate C3 and the first filter residue D3, and the first filter residue D3 was washed with acetone for several times to remove some simple adsorbed non-glycine ingredients, while the first filtrate C3 was left to be used for the second extraction, the collected first filter residue D3 was placed into the beaker with deionized water, and ultrasound was performed for half an hour, then the second filtrate E3 and the second filter residue F3 were obtained through filtration, the second filter residue F3 was washed with hot water for several times, after drying and activating, it was left to be subsequent recycled, the second filtrate was decompressed to remove water, and the white crystal powder can be obtained, the glycine product can be obtained after the obtained white crystal powder was separated and dried, besides, the product can be further purified through recystallization.

(16) Step 5: stepwise separation of serine, comprising following steps:

(17) At first, carrying out pH adjustment, the pH value of the first filtrate C3 obtained in step 4 was adjusted to 3.23- 5.67 by repeating the pH adjustment, by this time, the serine was positively charged, while other amino acids were negatively charged;

(18) Next, carrying out serine selective extraction, some graphene nano materials were added into the second filtrate C3 and ultrasound was performed for half an hour, the positively charged serine was combined with the negatively charged graphene nano material assisted by ultrasonic wave, then the Camellia nitidissima Chi concentrated solution. B4 to be separated was formed.

(19) Then carrying out serine stepwise separation, the suction filtration as carried out to the Camellia nitidissima Chi concentrated solution B4 to obtain the first filtrate C4 and the first filter residue D4 and the first filter residue D4 was washed with acetone for several times to remove some simple adsorbed non-serine ingredients, while the first filtrate C4 was left to be used for the second extraction, the collected first filter residue D4 was placed into the beaker with 95% methanol, and ultrasound was performed for half an hour, then the second filtrate E4 and the second filter residue F4 were obtained through filtration, the second filter residue F4 was washed with methanol for several times, after drying and activating, it was left to be subsequent recycled, the methanol of the second filtrate E4 was removed by rotary evaporation, and the white crystal can be obtained, the serine product can be obtained after the obtained white crystal was separated and dried, the product can be further purified through recystallization.

(20) Step 6: stepwise separation of glutamic acid, comprising allowing steps:

(21) At first, carrying out pH adjustment, the pH value of the first filtrate C4 obtained in step 5 was adjusted to 2.78- 3.21 by repeating the pH adjustment, by this time, the glutamic acid was positively charged, while other amino acids were negatively charged;

(22) Next, carrying out glutamic acid selective extraction, some graphene nano materials were added into the second filtrate C4 and the mixed materials of unmodified graphene, graphene modified by chitosan and graphene modified by metal ions were taken as the graphene nano material, ultrasound was performed for half an hour, and the positively charged glutamic acid was combined with the negatively charged graphene nano material assisted by ultrasonic wave, then the Camellia nitidissima Chi concentrated solution. B5 to be separated was formed.

(23) Then carrying out glutamic acid stepwise separation, the suction filtration was carried out to the Camellia nitidissima Chi concentrated solution B5 to obtain the first filtrate C5 and the first filter residue D5, and the first filter residue D5 was washed with acetone for several times to remove some simple adsorbed non-glutamic acid ingredients, and the first filtrate C5 was left to be used for the second extraction, while the first filter residue D5 was placed into the beaker with hot water after collection, and performing ultrasound for half an hour, then the second filtrate E5 and the second filter residue F5 were obtained through immediate filtration, the second filter residue was washed with hot water for several times, after drying and activating, it was left to be subsequent recycled, the second filtrate was placed into the beaker and heated to high temperature, in a successive step, the beaker with the second filtrate E5 was placed into ice water mixture immediately, the threonine product can be obtained after the precipitated scaly crystal was separated and dried, besides, the product can be further purified through recystallization.

(24) Step 7: stepwise separation of aspartic acid, comprising following steps:

(25) At first, carrying out pH adjustment, the pH value of the first filtrate C5 obtained in step 6 was adjusted to be less than 2.77 by repeating the pH adjustment, by this time, the aspartic acid was positively charged.

(26) Next, carrying out aspartic acid selective extraction, some graphene nano materials were added into the first filtrate C5 and the mixed materials of graphene modified by biomacromolecule and graphene-like nanometer mesoporous material were taken as the graphene nano material, ultrasound was performed for half an hour, and the positively charged aspartic acid was combined with the negatively charged graphene nano material assisted by ultrasonic wave, then the Camellia nitidissima Chi concentrated solution B6 to be separated was obtained.

(27) Then carrying out aspartic acid stepwise separation, the suction filtration was carried out to the Camellia nitidissima Chi concentrated solution 86 to obtain the first filtrate C6 and the first filter residue D6, and the first filter residue D6 was washed with acetone for several times to remove some simple adsorbed non-aspartic acid ingredients, while the first filtrate C6 was left to be used for the extraction of other ingredients, the collected first filter residue D6 was placed into the beaker with hot water, and ultrasound was performed for half an hour, then the second filtrate E6 and the second residue F6 were obtained through immediate filtration, the second filter residue was washed with hot water for several times, after drying and activating, it was left to be subsequent recycled, the second filtrate was placed into the beaker and heated to high temperature, in a successive step, the beaker with the second filtrate E6 was placed into ice water mixture immediately, the threonine product can be obtained after the precipitated scaly crystal was separated and dried, besides, the product can be further purified through recystallization.

(28) The first filtrate E6 after removing the amino acid active ingredients contains rich active ingredients, such as tea polyphenols, linoleic acids, total flavones, tea polysaccharides, they can be used for the development of other products of Camellia nitidissima Chi through comprehensive utilization.

(29) To reduce the cost effectively, the graphene nano materials are synthesized by renewable resources derived active carbons with improved Hummers method and reversible addition-fragmentation chain transfer polymerization method. The raw materials to prepare active carbon by carbonizing renewable resources include straw, bagasse and cornstalk.

(30) The above disclosure merely shows several specific embodiments of the present invention, and the present invention is not limited thereto; those ordinary skilled in the art complete the implementation of the present invention without difficulty based on the description and above disclosure; while it should be noted to those skilled in the art that several variations, modification and improvements can also be made within the scope of technical proposal, and these variations, modification and improvements are equivalent embodiments; moreover, they are also considered within the protective scope of the present invention.