CN102675483B

CN102675483B - Homogeneous pectic polysaccharides and method for obtaining same from tea polysaccharides

Info

Publication number: CN102675483B
Application number: CN201210172141.2A
Authority: CN
Inventors: 王顺春; 王辉俊; 鲍斌; 施松善; 王峥涛; 胡之璧
Original assignee: Shanghai University of Traditional Chinese Medicine
Current assignee: Shanghai University of Traditional Chinese Medicine
Priority date: 2012-05-30
Filing date: 2012-05-30
Publication date: 2014-03-05
Anticipated expiration: 2032-05-30
Also published as: CN102675483A

Abstract

The invention discloses homogeneous pectic polysaccharides and a method for obtaining the same from tea polysaccharides. In particular, the invention relates to polygalacturonic acid homogeneous pectic polysaccharides and a method for obtaining the same from tea polysaccharides or tea leaf polysaccharides. The homogeneous pectic polysaccharides and the method have the following beneficial effects: 1,4-connected polygalacturonic acid pectic polysaccharides are obtained from tea polysaccharides through separation and purification for the first time; the preparation method is simple and is high in yield; acids, alkalies and other toxic and harmful reagents are not used in the whole preparation process, thus the stability of the activity structures of tea polysaccharides can be fully ensured and the preparation method is suitable for large-scale production; in particular, the tea leaves obtained after extracting tea polyphenols can be effectively utilized by using the method, thus the resources can be saved and the refuses can be reclaimed; and the method has great significance in study and production of polygalacturonic acid homogeneous pectic polysaccharides.

Description

A kind of method of homogeneous pectin polysaccharide and homogeneous pectin polysaccharide of acquisition of obtaining from tea polysaccharide

Technical field

The present invention relates to a kind of method of homogeneous pectin polysaccharide and homogeneous pectin polysaccharide of acquisition of obtaining from tea polysaccharide, specifically, relate to a kind of method of polygalacturonic acids homogeneous pectin polysaccharide and polygalacturonic acids homogeneous pectin polysaccharide of acquisition of obtaining from Tea Polysaccharides or tea grounds polysaccharide.

Background technology

Tealeaves is the bud-leaf of plant of theaceae.< < legendary god of farming book on Chinese herbal medicine > > records " tea is bitter and tremble with fear, and the yin aspect of yin, sinks and also fall also, can fall fire ".Be widespread use among the people.Modern medicine proves: tea has refrigerant, and removing toxic substances, quenches the thirst, and diuresis disappears and tiredly refreshes oneself, and the fat that disappears goes greasy, clears away heart-fire and soothes the spirit, the pharmacological effect such as promote longevity.Along with the progress of medical sci-tech, to tea component, the further investigation of pharmaceutical use, finds approximately to contain 500 Multiple components in tealeaves, has the kind more than 250 that has of pharmaceutical use.Tea polysaccharide be from tealeaves, extract there is multiple biological activity and baroque polysaccharide compound, be the another important physiologically active substance of finding after tealeaves relaying tea-polyphenol.From Japan Report in 1986 since Tea Polysaccharides has hypoglycemic activity, find that successively Tea Polysaccharides has the multiple biological activitys such as hypoglycemic, reducing blood-fat, radiation protection, anticoagulation, antithrombotic, anti-oxidant, enhancing body immunologic function, thereby make the research and development of tea polysaccharide become the focus of current research.

Research about tea polysaccharide relates generally to following content: a kind of method that discloses separation and Extraction natural tea polysaccharide in the Chinese patent literature that application number is 201010141786.0, adopt the adsorption column of inorganic zeolite molecular sieve to remove tea-polyphenol, caffeine and partial pigment, then add protein precipitant to remove albumen, after ultrafiltration, obtain tea polysaccharide; Application number is a kind of method of comprehensive separation and Extraction tea polysaccharide, theanine, tea-polyphenol and caffeine that discloses in 201010141764.4 Chinese patent literature, tea solution, after zeolite molecular sieve, obtains tea polysaccharide, theanine, tea-polyphenol and caffeine crude product with water, ammonia soln, ethanolic soln wash-out successively; Application number is a kind of method of separating sweet tea polysaccharide by ultrafiltration membranes that discloses in 200910244874.0 Chinese patent literature, adopts the separated tea polysaccharide of ultrafiltration process; Application number is in 200910068255.0 Chinese patent literature, to disclose a kind of method of extracting tea polysaccharide and tea-polyphenol from thick old green tea, use alcohol precipitation, and ultrafiltration and the CTAB precipitator method are prepared tea polysaccharide extract; Application number is in the Chinese patent literature of 200810106962.X, to disclose a kind of preparation method for separating and purifying and hypoglycemic activity of tea polysaccharide, adopt the extraction of microwave-assisted solvent, vacuum enzyme carry, radially high efficiency chromatography separated tea polysaccharide; Application number is in 200810106964.9 Chinese patent literature, to disclose a kind of preparation method for separating and purifying and Structural Identification of tea polysaccharide, adopt the extraction of microwave-assisted solvent, vacuum enzyme carry, radially high efficiency chromatography separated thick tea polysaccharide carried out simple structural analysis; Application number is in 200810106963.4 Chinese patent literature, to disclose a kind of preparation method for separating and purifying and anti-tumor activity of tea polysaccharide, adopt equally the extraction of microwave-assisted solvent, vacuum enzyme carry, radially high efficiency chromatography separated tea polysaccharide screen its anti-tumor activity; Application number is in 200710037543.0 Chinese patent literature, to disclose a kind of preparation technology of high purity tea polysaccharide, and tealeaves soaks and removes the impurity such as polyphenol with enzyme solution or organic solvent, after ion-exchange resin decolorization deproteinated; Application number is to disclose a kind of method of extracting tea-polyphenol by-product trimethyl-xanthine and tea polysaccharide from tealeaves in 200510042629.3 Chinese patent literature, adopts complex enzyme hydrolysis to extract and water extraction tea-polyphenol, trimethyl-xanthine and tea polysaccharide; Application number is in 03114898.0 Chinese patent literature, to disclose a kind of method of extracting tea polysaccharide from extracted the tankage of tea-polyphenol, by adding polygalacturonase, extracts tea polysaccharide; Application number is to disclose a kind of method of extracting tea-polyphenol and byproduct thereof in tealeaves in 96113134.9 Chinese patent literature; Application number is in 97109115.3 Chinese patent literature, to disclose a kind ofly from plain tea leaves and old tea leaves, to extract polysaccharide, polyphenol mixed crystal processing method; Application number is in 01134065.7 Chinese patent literature, to disclose a kind of extracting method of tea polysaccharide; Application number is to disclose a kind of purifying tea polysaccharide and deduction method in 02110999.0 Chinese patent literature, adds albumen remover and reach purifying object except impurity such as albumen after water extraction; Application number be in 200410015905.2 Chinese patent literature, disclose a kind of from tealeaves the comprehensive method of extracting tea polysaccharide, tea-polyphenol, theanine, trimethyl-xanthine, under microwave action, repeatedly repeat to soak and extract tea polysaccharide; Application number is to disclose a kind of effective content of tea comprehensive preparation technology in 200310114694.3 Chinese patent literature; Application number is to disclose the method for extracting tea-polyphenol, theanine, tea polysaccharide, tea pigment from tealeaves in 200610055145.7 Chinese patent literature; Application number is to disclose a kind of tea polysaccharide and its production and use in 200610125174.6 Chinese patent literature, after water extract-alcohol precipitation, precipitates water-solublely and with different ultra-filtration membrane ultrafiltration, by different molecular weight, holds back separation.

The content to sum up studied is visible, is mainly obtaining and activity research aspect of tea polysaccharide at present to the research of tea polysaccharide, and wherein the acquisition methods about tea polysaccharide mainly contains water extraction and alcohol precipitation method, acidleach formulation, alkali method, Enzymatic Extraction method; But wherein acidleach formulation and alkali method are easy to make part tea polysaccharide hydrolysis, thereby destroy the active structure of tea polysaccharide, and Enzymatic Extraction method not only cost is higher but also can reduce the tea polysaccharide yield containing uronic acid part; And majority also relates to poisonous harmful reagent in disclosed purification technique.Although contain polygalacturonic acids pectin polysaccharide in research report tea polysaccharide, how from tea polysaccharide, separation and purification obtains polygalacturonic acids homogeneous pectin polysaccharide, so far there are no any correlation technique report.

Summary of the invention

The problems referred to above and the defect that for prior art, exist, the object of this invention is to provide a kind of method of polygalacturonic acids homogeneous pectin polysaccharide and polygalacturonic acids homogeneous pectin polysaccharide of acquisition of obtaining from Tea Polysaccharides or tea grounds polysaccharide, for research and the production of polygalacturonic acids homogeneous pectin polysaccharide provides a new way.

For achieving the above object, the technical solution used in the present invention is as follows:

A method of obtaining homogeneous pectin polysaccharide from tea polysaccharide, comprises the steps:

A) the dry tea grounds after dry tealeaves or extraction tea-polyphenol is crushed to 40～100 orders;

B) water extracts secondaries in 100 ℃, adds for the first time volume of water and be tealeaves or tea grounds quality 15 times, adds for the second time volume of water and be tealeaves or tea grounds quality 10 times; Merge secondary raffinate, concentrated, add ethanol to carry out alcohol precipitation, the alcohol concn of controlling in alcohol precipitation system is 40vol% or 70vol%; After alcohol precipitation 24 hours, filter collecting precipitation; Precipitation is carried out to centrifugal and lyophilize, obtain Tea Polysaccharides crude product or tea grounds polysaccharide crude;

C) by step b) the Tea Polysaccharides crude product or the tea grounds polysaccharide crude that obtain be splined on DEAE-Sepharose post, and the sodium chloride aqueous solution of water, 0.1mol/L is, the sodium chloride aqueous solution of 0.2mol/L carries out wash-out successively; Use sulfuric acid-phynol method to follow the tracks of elution curve, collect and utilize the sodium chloride aqueous solution of 0.1mol/L and the sodium chloride aqueous solution of 0.2mol/L to carry out the efficient part of wash-out respectively;

D) by step c) efficient part that obtains dissolves with the sodium chloride aqueous solution of suitable quantity of water or 0.2mol/L respectively, centrifugal, get supernatant liquor and be splined on propylene sephadex chromatography post or sephadex chromatography post, the sodium chloride aqueous solution of water or 0.2mol/L carries out wash-out; Utilize differential refraction detector to detect and follow the tracks of elution curve, according to elution curve, collect the efficient part of wash-out; Concentrated, lyophilize, obtains homogeneous pectin polysaccharide: be the polygalacturonic acids pectin polysaccharide that Isosorbide-5-Nitrae connects.

By aforesaid method can obtain in following homogeneous pectin polysaccharide any one or more than two kinds:

Molecular-weight average is 2.7 * 10 ⁵dalton, galactouronic acid content is 90.7%, and esterification degree is 28.0%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 1.97 ‰ Isosorbide-5-Nitrae connects, and is designated as TPS4-1B;

Molecular-weight average is 3.1 * 10 ⁴dalton, galactouronic acid content is 100%, and esterification degree is 28.4%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 4.84 ‰ Isosorbide-5-Nitrae connects, and is designated as TPS4-2A;

Molecular-weight average is 1.0 * 10 ⁴dalton, galactouronic acid content is 99.5%, and esterification degree is 35.0%, and the polygalacturonic acids pectin polysaccharide that the Isosorbide-5-Nitrae that acetylation degree is 0 connects, is designated as TPS7-1B;

Molecular-weight average is 6.5 * 10 ⁴dalton, galactouronic acid content is 87.6%, and esterification degree is 9.5%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 57.2 ‰ Isosorbide-5-Nitrae connects, and is designated as TPS7-2A;

Molecular-weight average is 8.6 * 10 ⁵dalton, galactouronic acid content is 99.0%, and esterification degree is 30.5%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 1.27 ‰ Isosorbide-5-Nitrae connects, and is designated as TPSR4-1B;

Molecular-weight average is 3.7 * 10 ⁵dalton, galactouronic acid content is 98.5%, and esterification degree is 28.3%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 9.15 ‰ Isosorbide-5-Nitrae connects, and is designated as TPSR4-2A;

Molecular-weight average is 6.5 * 10 ⁴dalton, galactouronic acid content is 100%, and esterification degree is 33.3%, and the polygalacturonic acids pectin polysaccharide that the Isosorbide-5-Nitrae that acetylation degree is 0 connects, is designated as TPSR7-1B;

Molecular-weight average is 2.8 * 10 ⁵dalton, galactouronic acid content is 100%, and esterification degree is 26.1%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 7.97 ‰ Isosorbide-5-Nitrae connects, and is designated as TPSR7-2A.

A preparation for described TPS4-1B homogeneous pectin polysaccharide, comprises the steps:

11) will be dried tea leaf powder and be broken to 40～100 orders;

12) water extracts secondaries in 100 ℃, adds for the first time volume of water and be 15 times of tea quality, adds for the second time volume of water and be 10 times of tea quality; Merge secondary raffinate, concentrated, add ethanol to carry out alcohol precipitation, the alcohol concn of controlling in alcohol precipitation system is 40vol%; After alcohol precipitation 24 hours, filter collecting precipitation; Precipitation is carried out to centrifugal and lyophilize, obtain Tea Polysaccharides crude product;

13) by step 12) the Tea Polysaccharides crude product that obtains is splined on DEAE-Sepharose post, and the sodium chloride aqueous solution of water, 0.1mol/L carries out wash-out successively; Use sulfuric acid-phynol method to follow the tracks of elution curve, collection utilizes the sodium chloride aqueous solution of 0.1mol/L to carry out the efficient part of wash-out;

14) by step 13) efficient part that obtains dissolves with the sodium chloride aqueous solution of appropriate 0.2mol/L, centrifugal, gets supernatant liquor and is splined on propylene sephadex chromatography post, with the sodium chloride aqueous solution of 0.2mol/L, carries out wash-out; Utilize differential refraction detector to detect and follow the tracks of elution curve, according to elution curve, collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide.

A preparation for described TPS4-2A homogeneous pectin polysaccharide, comprises the steps:

21) will be dried tea leaf powder and be broken to 40～100 orders;

22) water extracts secondaries in 100 ℃, adds for the first time volume of water and be 15 times of tea quality, adds for the second time volume of water and be 10 times of tea quality; Merge secondary raffinate, concentrated, add ethanol to carry out alcohol precipitation, the alcohol concn of controlling in alcohol precipitation system is 40vol%; After alcohol precipitation 24 hours, filter collecting precipitation; Precipitation is carried out to centrifugal and lyophilize, obtain Tea Polysaccharides crude product;

23) by step 22) the Tea Polysaccharides crude product that obtains is splined on DEAE-Sepharose post, and the sodium chloride aqueous solution of water, 0.1mol/L and the sodium chloride aqueous solution of 0.2mol/L carry out wash-out successively; Use sulfuric acid-phynol method to follow the tracks of elution curve, collection utilizes the sodium chloride aqueous solution of 0.2mol/L to carry out the efficient part of wash-out;

24) by step 23) efficient part that obtains dissolves by suitable quantity of water, centrifugal, gets supernatant liquor and is splined on sephadex chromatography post, and water carries out wash-out; Utilize differential refraction detector to detect and follow the tracks of elution curve, according to elution curve, collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide.

A preparation for described TPS7-1B homogeneous pectin polysaccharide, comprises the steps:

31) will be dried tea leaf powder and be broken to 40～100 orders;

32) water extracts secondaries in 100 ℃, adds for the first time volume of water and be 15 times of tea quality, adds for the second time volume of water and be 10 times of tea quality; Merge secondary raffinate, concentrated, add ethanol to carry out alcohol precipitation, the alcohol concn of controlling in alcohol precipitation system is 70vol%; After alcohol precipitation 24 hours, filter collecting precipitation; Precipitation is carried out to centrifugal and lyophilize, obtain Tea Polysaccharides crude product;

33) by step 32) the Tea Polysaccharides crude product that obtains is splined on DEAE-Sepharose post, and the sodium chloride aqueous solution of water, 0.1mol/L carries out wash-out successively; Use sulfuric acid-phynol method to follow the tracks of elution curve, collection utilizes the sodium chloride aqueous solution of 0.1mol/L to carry out the efficient part of wash-out;

34) by step 33) efficient part that obtains dissolves with the sodium chloride aqueous solution of appropriate 0.2mol/L, centrifugal, gets supernatant liquor and is splined on propylene sephadex chromatography post, with the sodium chloride aqueous solution of 0.2mol/L, carries out wash-out; Utilize differential refraction detector to detect and follow the tracks of elution curve, according to elution curve, collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide.

A preparation for described TPS7-2A homogeneous pectin polysaccharide, comprises the steps:

41) will be dried tea leaf powder and be broken to 40～100 orders;

42) water extracts secondaries in 100 ℃, adds for the first time volume of water and be 15 times of tea quality, adds for the second time volume of water and be 10 times of tea quality; Merge secondary raffinate, concentrated, add ethanol to carry out alcohol precipitation, the alcohol concn of controlling in alcohol precipitation system is 70vol%; After alcohol precipitation 24 hours, filter collecting precipitation; Precipitation is carried out to centrifugal and lyophilize, obtain Tea Polysaccharides crude product;

43) by step 42) the Tea Polysaccharides crude product that obtains is splined on DEAE-Sepharose post, and the sodium chloride aqueous solution of water, 0.1mol/L and the sodium chloride aqueous solution of 0.2mol/L carry out wash-out successively; Use sulfuric acid-phynol method to follow the tracks of elution curve, collection utilizes the sodium chloride aqueous solution of 0.2mol/L to carry out the efficient part of wash-out;

44) by step 43) efficient part that obtains dissolves by suitable quantity of water, centrifugal, gets supernatant liquor and is splined on sephadex chromatography post, and water carries out wash-out; Utilize differential refraction detector to detect and follow the tracks of elution curve, according to elution curve, collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide.

A preparation for described TPSR4-1B homogeneous pectin polysaccharide, comprises the steps:

51) the dry tea grounds extracting after tea-polyphenol is crushed to 40～100 orders;

52) water extracts secondaries in 100 ℃, adds for the first time volume of water and be 15 times of tea grounds quality, adds for the second time volume of water and be 10 times of tea grounds quality; Merge secondary raffinate, concentrated, add ethanol to carry out alcohol precipitation, the alcohol concn of controlling in alcohol precipitation system is 40vol%; After alcohol precipitation 24 hours, filter collecting precipitation; Precipitation is carried out to centrifugal and lyophilize, obtain tea grounds polysaccharide crude;

53) by step 52) the tea grounds polysaccharide crude that obtains is splined on DEAE-Sepharose post, and the sodium chloride aqueous solution of water, 0.1mol/L carries out wash-out successively; Use sulfuric acid-phynol method to follow the tracks of elution curve, collection utilizes the sodium chloride aqueous solution of 0.1mol/L to carry out the efficient part of wash-out;

54) by step 53) efficient part that obtains dissolves by suitable quantity of water, centrifugal, gets supernatant liquor and is splined on propylene sephadex chromatography post, and water carries out wash-out; Utilize differential refraction detector to detect and follow the tracks of elution curve, according to elution curve, collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide.

A preparation for described TPSR4-2A homogeneous pectin polysaccharide, comprises the steps:

61) the dry tea grounds extracting after tea-polyphenol is crushed to 40～100 orders;

62) water extracts secondaries in 100 ℃, adds for the first time volume of water and be 15 times of tea grounds quality, adds for the second time volume of water and be 10 times of tea grounds quality; Merge secondary raffinate, concentrated, add ethanol to carry out alcohol precipitation, the alcohol concn of controlling in alcohol precipitation system is 40vol%; After alcohol precipitation 24 hours, filter collecting precipitation; Precipitation is carried out to centrifugal and lyophilize, obtain tea grounds polysaccharide crude;

63) by step 62) the tea grounds polysaccharide crude that obtains is splined on DEAE-Sepharose post, and the sodium chloride aqueous solution of water, 0.1mol/L and the sodium chloride aqueous solution of 0.2mol/L carry out wash-out successively; Use sulfuric acid-phynol method to follow the tracks of elution curve, collection utilizes the sodium chloride aqueous solution of 0.2mol/L to carry out the efficient part of wash-out;

64) by step 63) efficient part that obtains dissolves by suitable quantity of water, centrifugal, gets supernatant liquor and is splined on sephadex chromatography post, and water carries out wash-out; Utilize differential refraction detector to detect and follow the tracks of elution curve, according to elution curve, collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide.

A preparation for described TPSR7-1B homogeneous pectin polysaccharide, comprises the steps:

71) the dry tea grounds extracting after tea-polyphenol is crushed to 40～100 orders;

72) water extracts secondaries in 100 ℃, adds for the first time volume of water and be 15 times of tea grounds quality, adds for the second time volume of water and be 10 times of tea grounds quality; Merge secondary raffinate, concentrated, add ethanol to carry out alcohol precipitation, the alcohol concn of controlling in alcohol precipitation system is 70vol%; After alcohol precipitation 24 hours, filter collecting precipitation; Precipitation is carried out to centrifugal and lyophilize, obtain tea grounds polysaccharide crude;

73) by step 72) the tea grounds polysaccharide crude that obtains is splined on DEAE-Sepharose post, and the sodium chloride aqueous solution of water, 0.1mol/L carries out wash-out successively; Use sulfuric acid-phynol method to follow the tracks of elution curve, collection utilizes the sodium chloride aqueous solution of 0.1mol/L to carry out the efficient part of wash-out;

74) by step 73) efficient part that obtains dissolves by suitable quantity of water, centrifugal, gets supernatant liquor and is splined on propylene sephadex chromatography post, and water carries out wash-out; Utilize differential refraction detector to detect and follow the tracks of elution curve, according to elution curve, collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide.

A preparation for described TPSR7-2A homogeneous pectin polysaccharide, comprises the steps:

81) the dry tea grounds extracting after tea-polyphenol is crushed to 40～100 orders;

82) water extracts secondaries in 100 ℃, adds for the first time volume of water and be 15 times of tea grounds quality, adds for the second time volume of water and be 10 times of tea grounds quality; Merge secondary raffinate, concentrated, add ethanol to carry out alcohol precipitation, the alcohol concn of controlling in alcohol precipitation system is 70vol%; After alcohol precipitation 24 hours, filter collecting precipitation; Precipitation is carried out to centrifugal and lyophilize, obtain tea grounds polysaccharide crude;

83) by step 82) the tea grounds polysaccharide crude that obtains is splined on DEAE-Sepharose post, and the sodium chloride aqueous solution of water, 0.1mol/L and the sodium chloride aqueous solution of 0.2mol/L carry out wash-out successively; Use sulfuric acid-phynol method to follow the tracks of elution curve, collection utilizes the sodium chloride aqueous solution of 0.2mol/L to carry out the efficient part of wash-out;

84) by step 83) efficient part that obtains dissolves with the sodium chloride aqueous solution of appropriate 0.2mol/L, centrifugal, gets supernatant liquor and is splined on sephadex chromatography post, with the sodium chloride aqueous solution of 0.2mol/L, carries out wash-out; Utilize differential refraction detector to detect and follow the tracks of elution curve, according to elution curve, collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide.

Compared with prior art, the present invention first from tea polysaccharide separation and purification obtained and be 1, the 4 polygalacturonic acids pectin polysaccharides that connect, and preparation method is simple, yield is high, in whole preparation process, do not use acid, alkali and other poisonous and harmful reagent, can fully guarantee the stability of tea polysaccharide active structure, be applicable to large-scale production; Especially, use the inventive method can effectively utilize the tea grounds extracting after tea-polyphenol, can economize on resources, realize refuse reclamation, to the research of polygalacturonic acids homogeneous pectin polysaccharide with produce significant.

Accompanying drawing explanation

Fig. 1-1st, embodiment 1 obtains the gel chromatography elution curve of homogeneous pectin polysaccharide TPS4-1B;

Fig. 1-2 is high performance liquid phase gel chromatography (HPGPC) figure of the homogeneous pectin polysaccharide TPS4-1B that obtains of embodiment 1;

Fig. 1-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPS4-1B that embodiment 1 obtains after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize;

Fig. 1-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPS4-1B that embodiment 1 obtains;

Fig. 1-5th, the GC-MS after the homogeneous pectin polysaccharide TPS4-1B that embodiment 1 obtains methylates analyzes collection of illustrative plates;

Fig. 2-1st, embodiment 2 obtains the elution curve of gel chromatography for the first time of homogeneous pectin polysaccharide TPS4-2A;

Fig. 2-2nd, embodiment 2 obtains the elution curve of gel chromatography for the second time of homogeneous pectin polysaccharide TPS4-2A;

Fig. 2-3rd, high performance liquid phase gel chromatography (HPGPC) figure of the homogeneous pectin polysaccharide TPS4-2A that embodiment 2 obtains;

Fig. 2-4th, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPS4-2A that embodiment 2 obtains after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize;

Fig. 2-5th, the carbon spectrogram of the homogeneous pectin polysaccharide TPS4-2A that embodiment 2 obtains;

Fig. 2-6th, the GC-MS after the homogeneous pectin polysaccharide TPS4-2A that embodiment 2 obtains methylates analyzes collection of illustrative plates;

Fig. 3-1st, embodiment 3 obtains the gel chromatography elution curve of homogeneous pectin polysaccharide TPS7-1B;

Fig. 3-2nd, high performance liquid phase gel chromatography (HPGPC) figure of the homogeneous pectin polysaccharide TPS7-1B that embodiment 3 obtains;

Fig. 3-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPS7-1B that embodiment 3 obtains after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize;

Fig. 3-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPS7-1B that embodiment 3 obtains;

Fig. 3-5th, the GC-MS after the homogeneous pectin polysaccharide TPS7-1B that embodiment 3 obtains methylates analyzes collection of illustrative plates;

Fig. 4-1st, embodiment 4 obtains the gel chromatography elution curve of homogeneous pectin polysaccharide TPS7-2A;

Fig. 4-2nd, high performance liquid phase gel chromatography (HPGPC) figure of the homogeneous pectin polysaccharide TPS7-2A that embodiment 4 obtains;

Fig. 4-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPS7-2A that embodiment 4 obtains after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize;

Fig. 4-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPS7-2A that embodiment 4 obtains;

Fig. 4-5th, the GC-MS after the homogeneous pectin polysaccharide TPS7-2A that embodiment 4 obtains methylates analyzes collection of illustrative plates;

Fig. 5-1st, embodiment 5 obtains the gel chromatography elution curve of homogeneous pectin polysaccharide TPSR4-1B;

Fig. 5-2nd, high performance liquid phase gel chromatography (HPGPC) figure of the homogeneous pectin polysaccharide TPSR4-1B that embodiment 5 obtains;

Fig. 5-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPSR4-1B that embodiment 5 obtains after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize;

Fig. 5-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPSR4-1B that embodiment 5 obtains;

Fig. 5-5th, the GC-MS after the homogeneous pectin polysaccharide TPSR4-1B that embodiment 5 obtains methylates analyzes collection of illustrative plates;

Fig. 6-1st, embodiment 6 obtains the gel chromatography elution curve of homogeneous pectin polysaccharide TPSR4-2A;

Fig. 6-2nd, high performance liquid phase gel chromatography (HPGPC) figure of the homogeneous pectin polysaccharide TPSR4-2A that embodiment 6 obtains;

Fig. 6-3rd, the GC collection of illustrative plates comparison diagram of the homogeneous pectin polysaccharide TPSR4-2A that embodiment 6 obtains before and after acetylize;

Fig. 6-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPSR4-2A that embodiment 6 obtains;

Fig. 6-5th, the GC-MS after the homogeneous pectin polysaccharide TPSR4-2A that embodiment 6 obtains methylates analyzes collection of illustrative plates;

Fig. 7-1st, embodiment 7 obtains the gel chromatography elution curve of homogeneous pectin polysaccharide TPSR7-1B;

Fig. 7-2nd, high performance liquid phase gel chromatography (HPGPC) figure of the homogeneous pectin polysaccharide TPSR7-1B that embodiment 7 obtains;

Fig. 7-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPSR7-1B that embodiment 7 obtains after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize;

Fig. 7-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPSR7-1B that embodiment 7 obtains;

Fig. 7-5th, the GC-MS after the homogeneous pectin polysaccharide TPSR7-1B that embodiment 7 obtains methylates analyzes collection of illustrative plates;

Fig. 8-1st, embodiment 8 obtains the gel chromatography elution curve of homogeneous pectin polysaccharide TPSR7-2A;

Fig. 8-2nd, high performance liquid phase gel chromatography (HPGPC) figure of the homogeneous pectin polysaccharide TPSR7-2A that embodiment 8 obtains;

Fig. 8-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPSR7-2A that embodiment 8 obtains after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize;

Fig. 8-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPSR7-2A that embodiment 8 obtains;

Fig. 8-5th, the GC-MS after the homogeneous pectin polysaccharide TPSR7-2A that embodiment 8 obtains methylates analyzes collection of illustrative plates.

Embodiment

Below in conjunction with embodiment to the present invention do further in detail, intactly explanation.The experimental technique of unreceipted actual conditions in following embodiment, conventionally according to normal condition or the condition of advising according to manufacturer.

Embodiment 1: prepare homogeneous pectin polysaccharide TPS4-1B

11) will be dried tea leaf powder and be broken to 40～100 orders;

14) by step 13) efficient part that obtains dissolves with the sodium chloride aqueous solution of appropriate 0.2mol/L, centrifugal, get supernatant liquor and be splined on propylene sephadex chromatography post Sephacryl S-300 High Resolution, with the sodium chloride aqueous solution of 0.2mol/L, carry out wash-out; Utilize Shodex R1-102 differential refraction detector to detect and follow the tracks of elution curve, according to elution curve (seeing shown in Fig. 1-1), collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide TPS4-1B, total yield: 0.74wt%.

One, molecular weight determination

Adopt high performance liquid phase gel chromatography (HPGPC) method: chromatographic column is KS-805 and KS-804 series connection; Moving phase is the sodium chloride aqueous solution of 0.2mol/L; Column temperature: 40 ℃; Flow velocity: 0.8mL/min; Sampling volume: 20 μ L.

Get the DextranP-series standard dextran P-5 that molecular weight is known, P-10, P-20, P-50, P-100, P-200, each 2mg of P-400 and P-800, is mixed with respectively the solution of 2mg/mL with the sodium chloride aqueous solution of moving phase 0.2mol/L; With the centrifugal 10min of 10000rpm; Get supernatant liquor, each sample introduction 20 μ L; Logarithmic value with molecular weight is done typical curve to retention time.

Get testing sample 2mg, with the sodium chloride aqueous solution of moving phase 0.2mol/L, be mixed with the solution of 2mg/mL; With the centrifugal 10min of 10000rpm; Get supernatant liquor, sample introduction 20 μ L, carry out the test of high performance liquid phase gel chromatography.

According to the sample retention time of measuring, can from typical curve, try to achieve the molecular-weight average of sample.

The drafting processing of typical curve and molecular weight calculate and are automatically completed by GPC software.

Fig. 1-2 is high performance liquid phase gel chromatography (HPGPC) figure of the homogeneous pectin polysaccharide TPS4-1B that obtains, can be illustrated: the polysaccharide obtaining is homogeneous polysaccharide by Fig. 1-2.

Through measuring and calculating, the molecular-weight average of the homogeneous pectin polysaccharide TPS4-1B obtaining is 2.7 * 10 ⁵dalton.

Two, Structural Identification

1) completely acid hydrolysis and tlc analysis: get 2mg TPS4-1B sample, be placed in peace and cut open bottle, add 2M TFA 4mL, sealing, 121 ℃ of hydrolysis 2h, are hydrolyzed completely, repeatedly add methyl alcohol evaporate to dryness and remove TFA.Then add 0.1mL distilled water sample dissolution, on PEI-cellulose plate, contrast and carry out TLC analysis with standard monose, the kind of monosaccharide residue in rough determination polysaccharide, and judge whether containing uronic acid and hydrolysis reaction whether completely.Developping agent is ethyl acetate: pyridine: water: and acetic acid (5: 5: 3: 1, v/v).Developer is aniline-phthalic acid, after sprinkling, in 100 ℃ of heating 5min, develops the color.TLC analyzes and shows that TPS4-1B is acidic polysaccharose.

2) reducing sugar aldehydic acid: get 100mg TPS4-1B sample, be dissolved in 20mL distilled water, be stirred to completely and dissolve; Add 200mg carbodiimide (CMC), stir, with the aqueous hydrochloric acid of 0.1mol/L, adjust the pH=4.75 of system, be controlled under pH=4.75 stirring reaction 3 hours; The NaBH that dropwise adds the 2mol/L of new preparation ₄aqueous solution 200mL, the aqueous hydrochloric acid that simultaneously drips 3mol/L makes the pH value of system remain on neutrality, and dropping process approximately needs 2h; Drip and finish the rear stirring 10 minutes that continues; Reaction solution is proceeded in dialysis tubing, carry out flowing water dialysis 48 hours; Use again distill water dialysis one day; Lyophilize; After freeze-drying, repeat as stated above again 2 times.

3) sample after uronic acid reduction is carried out to complete acid hydrolysis, method is referring to 1); Then carry out acetylize: the sample after acid hydrolysis adds methyl alcohol evaporate to dryness three times completely, and repetitive operation is to eliminate TFA completely; After hydrolysis, resistates adds 50mg NaBH ₄with 2mL water, room temperature reduction reaction is spent the night; Acetic acid reaction on the rocks is to remove excessive NaBH ₄on Rotary Evaporators, be concentrated into thick liquid, add methyl alcohol-Glacial acetic acid (5: 1) 3-5mL, evaporate to dryness three times, then add twice of methyl alcohol evaporate to dryness, obtain white powder, put into 105 ℃ of 15min of baking oven to remove moisture, add 3mL diacetyl oxide, acetylization reaction 1h in 101 ℃ of baking ovens, take out, repeatedly add 50 ℃ of water-bath decompression azeotropic evaporates to dryness of toluene to Powdered; Add suitable quantity of water and dissolve, add chloroform extraction three times, combined chloroform layer, washes with water three times, separates chloroform layer, with anhydrous sodium sulfate drying, after concentrating, carries out GC-MS analysis.

Fig. 1-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPS4-1B obtaining after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize; In b figure, be followed successively by from left to right rhamnosyl, Fucose, pectinose, wood sugar, seminose, glucose, semi-lactosi characteristic of correspondence peak; A figure is more known with b figure: in the GC collection of illustrative plates of the homogeneous pectin polysaccharide TPS4-1B obtaining after acetylize, to only have semi-lactosi characteristic peak, thereby can infer that TPS4-1B is comprised of galacturonic acid.

4) carbon spectrum analysis: TPS4-1B sample is dissolved with heavy water, and concentration is 0.1mg/mL, carries out ¹³c-NMR analyzes;

Fig. 1-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPS4-1B obtaining; From Fig. 1-4: in this polysaccharide, containing neutral sugar, (60-62ppm interval is not there are no CH ₂signal), forming result with monose coincide; 176 and 172ppm be respectively galacturonic acid carboxyl carbon signal and galacturonic acid methyl esters carboxyl carbon signal, anomeric carbon is positioned at 100-101ppm, show that it is configured as α configuration, 79-80ppm is 4 carbon signals of α-D galacturonic acid (and methyl esters), 69-72ppm is C2, C3 and C5 signal, 54ppm is methyl esters carbon signal.

5) methylation analysis: get the sample of 5mg after uronic acid reduction, be placed in 25mL round-bottomed flask, through P ₂o ₅dried overnight, then adds dry DMSO 3mL, then adds sodium hydroxide powder 500mg, airtight stirring 1 hour; Drip methyl iodide 0.2mL, about 15min drips off, and drips complete stirring reaction 20min; Drip again methyl iodide 0.5mL, drip complete stirring reaction 20min; Continue to drip methyl iodide 0.5mL, drip complete stirring reaction 1 hour; Add water 2mL termination reaction, add chloroform 3mL extraction, chloroform layer washes with water 3 times, and evaporate to dryness is removed chloroform; Resistates is hydrolyzed 2 hours with trifluoroacetic acid (TFA) aqueous solution of 2mol/L in 120 ℃; Evaporate to dryness, then adds 100mg sodium borohydride and 2mL water, and reduction reaction is spent the night, and adds after Glacial acetic acid destroys unnecessary sodium borohydride and adds after the repeated multiple times evaporate to dryness of methyl alcohol/Glacial acetic acid (volume ratio is 5: 1), adds aceticanhydride in 100 ℃ of acetylizes 1 hour; Add 2mL water and sodium bicarbonate powder that aceticanhydride is decomposed, then use chloroform extraction, water washing, anhydrous sodium sulfate drying; Carry out GC-MS analysis;

Fig. 1-5th, the GC-MS after the homogeneous pectin polysaccharide TPS4-1B obtaining methylates analyzes collection of illustrative plates; A figure is wherein the GC figure after homogeneous pectin polysaccharide TPS4-1B methylates, and b figure is the corresponding mass spectrum in 27.81min peak in GC figure, and c figure is the corresponding mass spectrum in 32.02min peak in GC figure; In sample from Fig. 1-5: TPS4-1B after uronic acid reduction, only contain end group semi-lactosi (27.8min) and 1,4 semi-lactosis (32.0min) that connect, show that the sample of TPS4-1B after uronic acid reduction is straight chain 1,4 Polygalactans that connect, thereby further show that TPS4-1B is the polygalacturonic acid that straight chain Isosorbide-5-Nitrae connects.

6) periodate oxidation analysis: accurately take TPS4-1B sample 50mg, add 50mL 15 ~ 20mmol/LNaIO ₄the aqueous solution, under 4-20 ℃ of condition, lucifuge is placed, or jolting, in 4h, 24h, 48h ... interval time, sampling, diluted after 250 times, used uv-spectrophotometric instrument at 233nm place, to measure absorbance A, along with NaIO ₄consume, absorbance declines gradually until reach a stationary value, will record absorbance and the former NaIO of sample solution ₄the absorbance comparison of solution;

Analytical results shows: in sample TPS4-1B, the glycosyl of 1mol consumes the NaIO of 0.97mol ₄, show that its mode of connection is that Isosorbide-5-Nitrae connects.

Three, galactouronic acid assay

The aqueous sodium hydroxide solution compound concentration that the concentration of take is 0.5wt% is hydroxyl biphenyl test solution 10mL as between 0.15wt%, stand-by;

Accurately take 238.4mg sodium tetraborate decahydrate, sodium tetraborate decahydrate-sulfuric acid test solution 50mL that the concentrated sulfuric acid solution compound concentration that is 98% by concentration is 0.0125mol/L, stand-by;

Accurately take each 2.53mg of galacturonic acid standard substance and testing sample, with 25mL water, be made into galacturonic acid standardized solution (101.2 μ g/mL) and sample solution respectively;

Specification Curve of Increasing: get 6 clean Boiling tubes, add successively 0 μ L, 50 μ L, 100 μ L, 150 μ L, 200 μ L, 250 μ L galacturonic acid standardized solution, mend and add water to 250 μ L, ice bath is cooling, add 1.5mL sodium tetraborate decahydrate-sulfuric acid test solution, shake up, boiling water insulation 5 minutes, take out, ice bath is cooled to after room temperature, adds hydroxyl biphenyl test solution between 25 μ L, after shaking up with micropipette, at 520nm place, measure photoabsorption, with 0 pipe, do blank, drawing standard curve: Y=32.451X-0.012, R2=0.999.

Draw respectively sample solution 200 μ L for parallel 3 groups, constant volume is to 250 μ L, and all the other operations are prepared with typical curve.Measure absorbance value, by typical curve, calculate content.

Through measuring and calculating, the galactouronic acid content in the homogeneous pectin polysaccharide TPS4-1B obtaining is 90.7%.

Four, carry out esterification and acetylation analysis

1) key instrument

GC-MS:Thermo TRACE DSQ, attached fid detector;

Chromatographic column and chromatographic condition: DB-624(30m * 0.32mm * 1.8um, Agilent), the column temperature that carries out esterification assay is 65 ℃, and the column temperature that carries out acetylize assay is 100 ℃, and injector temperature is 200 ℃, and detector temperature is 320 ℃; Carrier gas is high pure nitrogen, and flow velocity is 2.0mL/min, and splitting ratio is 5: 1, and sampling volume is 0.1 μ L;

2) sample preparation

Precision takes 2.00mg testing sample to 10mL EP pipe, every pipe adds 1.2mL ultrapure water, airtight, the ultrasonic 10min of room temperature, adding 0.4mL concentration is the aqueous sodium hydroxide solution of 2mol/L, and in 25 ℃ of placement 1h, adding 0.4mL concentration is the aqueous hydrochloric acid of 2mol/L, stop saponification reaction, adjust pH to 2.0; Shake up, accurately pipette respectively 400 μ L and proceed to liquid phase bottle, respectively add 200 μ L100mg/L Virahol inner mark solutions, airtight to be measured, 4 parts of each sample horizontal surveies;

3) standardized solution builds

Respectively compound concentration be 20,40,60,80, methyl alcohol standard water solution and the acetic acid standard water solution of 100mg/L, and build typical curve with this concentration gradient.The concentration of standardized solution of take is respectively X-coordinate, and take the peak area of standardized solution and the peak area ratio of Virahol inner mark solution is ordinate zou, obtains regression equation: methyl alcohol, Y=0.0089X+0.0369, R ²=0.9981; Acetic acid, Y=0.0052X+0.0574, R ²=0.9935.

4) sample determination

With esterification and the acetylation content in set up GC-FID quantivative approach working sample, and calculate esterification degree (DS _first) and acetylation degree (DS _second): esterification degree is undertaken by formula, wherein W _first% is esterification content, DS _first≈ W _first% * 5.5; Acetylation degree is undertaken by formula, wherein W _second% is methyl esters content, DS second ≈ W _second% * M ₁/ 60;

Note: because esterification degree is larger, so need revise to average glycosyl molecular weight in polysaccharide chain M ₁for revised molecular weight, i.e. M ₁=190W _first+ (1-W _first) 176);

After measured, in the homogeneous pectin polysaccharide TPS4-1B obtaining, esterification content is 50.84mg/g, and esterification degree is 28.0%, and acetylation content is 2.32mg/g, and acetylation degree is 1.97 ‰.

Embodiment 2: prepare homogeneous pectin polysaccharide TPS4-2A

21) will be dried tea leaf powder and be broken to 40～100 orders;

22) water extracts secondaries in 100 ℃, adds for the first time volume of water and be 15 times of tea quality, adds for the second time volume of water and be 10 times of tea quality; Merge secondary raffinate, concentrated, add ethanol to carry out alcohol precipitation, the alcohol concn of controlling in alcohol precipitation system is 40vol%; Alcohol precipitation? after hour, filter collecting precipitation; Precipitation is carried out to centrifugal and lyophilize, obtain Tea Polysaccharides crude product;

24) by step 23) efficient part that obtains dissolves by suitable quantity of water, centrifugal, gets supernatant liquor and is splined on sephadex chromatography post Superdex200, and water carries out wash-out; Utilize Shodex R1-102 differential refraction detector to detect and follow the tracks of elution curve, according to elution curve (seeing shown in Fig. 2-1), collect the efficient part of wash-out; Again the efficient part of collecting is carried out to wash-out with the aqueous solution, utilize Shodex R1-102 differential refraction detector to detect and follow the tracks of elution curve, according to elution curve (seeing shown in Fig. 2-2), collect again the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide TPS4-2A, total yield: 6.80wt%.

High performance liquid phase gel chromatography (HPGPC) figure that Fig. 2-3 are the homogeneous pectin polysaccharide TPS4-2A that obtains, can be illustrated by Fig. 2-3: the polysaccharide obtaining is homogeneous polysaccharide.

With reference to the molecular weight determination measuring and calculating described in embodiment 1, learn that the molecular-weight average of obtained homogeneous pectin polysaccharide TPS4-2A is 3.1 * 10 ⁴dalton.

With reference to the Structural Identification content described in embodiment 1, obtained homogeneous pectin polysaccharide TPS4-2A is carried out to Structural Identification:

After the complete acid hydrolysis of TPS4-2A, TLC analyzes and shows that it is acidic polysaccharose, containing uronic acid;

Fig. 2-4th, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPS4-2A obtaining after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize; In b figure, be followed successively by from left to right rhamnosyl, Fucose, pectinose, wood sugar, seminose, glucose, semi-lactosi characteristic of correspondence peak; A figure is more known with b figure: in the GC collection of illustrative plates of the homogeneous pectin polysaccharide TPS4-2A obtaining after acetylize, to only have semi-lactosi characteristic peak, thereby can infer that TPS4-2A is comprised of galacturonic acid;

Fig. 2-5th, the carbon spectrogram of the homogeneous pectin polysaccharide TPS4-2A obtaining;

Fig. 2-6th, the GC-MS after the homogeneous pectin polysaccharide TPS4-2A obtaining methylates analyzes collection of illustrative plates, a figure is wherein the GC figure after homogeneous pectin polysaccharide TPS4-2A methylates, b figure is the corresponding mass spectrum in 27.81min peak in GC figure, and c figure is the corresponding mass spectrum in 32.00min peak in GC figure;

By structural analysis, learn: the polygalacturonic acids homogeneous pectin polysaccharide that the polysaccharide that the present embodiment obtains also connects for Isosorbide-5-Nitrae.

With reference to the galactouronic acid content assaying method described in embodiment 1, measuring and calculating learns that the galactouronic acid content in the homogeneous pectin polysaccharide that the present embodiment obtains is 100%.

With reference to the esterification described in embodiment 1 and acetylation analytical procedure, measure and to learn in the homogeneous pectin polysaccharide that the present embodiment obtains: esterification content is 51.57mg/g, and esterification degree is 28.4%; Acetylation content is 1.61mg/g, and acetylation degree is 4.84 ‰.

Embodiment 3: prepare homogeneous pectin polysaccharide TPS7-1B

31) will be dried tea leaf powder and be broken to 40～100 orders;

34) by step 33) efficient part that obtains dissolves with the sodium chloride aqueous solution of appropriate 0.2mol/L, centrifugal, get supernatant liquor and be splined on propylene sephadex chromatography post Sephacryl S-300 High Resolution, with the sodium chloride aqueous solution of 0.2mol/L, carry out wash-out; Utilize Shodex R1-102 differential refraction detector to detect and follow the tracks of the efficient part that elution curve (seeing shown in Fig. 3-1) is collected wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide TPS7-1B, total yield: 4.45wt%.

High performance liquid phase gel chromatography (HPGPC) figure that Fig. 3-2 are the homogeneous pectin polysaccharide TPS7-1B that obtains, can be illustrated by Fig. 3-2: the polysaccharide obtaining is homogeneous polysaccharide.

With reference to the molecular weight determination measuring and calculating described in embodiment 1, learn that the molecular-weight average of obtained homogeneous pectin polysaccharide TPS7-1B is 1.0 * 10 ⁴dalton.

With reference to the Structural Identification content described in embodiment 1, obtained homogeneous pectin polysaccharide TPS7-1B is carried out to Structural Identification:

After the complete acid hydrolysis of TPS7-1B, TLC analyzes and shows that it is acidic polysaccharose, containing uronic acid;

Fig. 3-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPS7-1B obtaining after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize; In b figure, be followed successively by from left to right rhamnosyl, Fucose, pectinose, wood sugar, seminose, glucose, semi-lactosi characteristic of correspondence peak; A figure is more known with b figure: in the GC collection of illustrative plates of the homogeneous pectin polysaccharide TPS7-1B obtaining after acetylize, to only have semi-lactosi characteristic peak, thereby can infer that TPS7-1B is comprised of galacturonic acid.

Fig. 3-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPS7-1B obtaining;

Fig. 3-5th, the GC-MS after the homogeneous pectin polysaccharide TPS7-1B obtaining methylates analyzes collection of illustrative plates, a figure is wherein the GC figure after homogeneous pectin polysaccharide TPS7-1B methylates, b figure is the corresponding mass spectrum in 27.38min peak in GC figure, and c figure is the corresponding mass spectrum in 31.50min peak in GC figure;

With reference to the galactouronic acid content assaying method described in embodiment 1, measuring and calculating learns that the galactouronic acid content in the homogeneous pectin polysaccharide that the present embodiment obtains is 99.5%.

With reference to the esterification described in embodiment 1 and acetylation analytical procedure, measure and to learn in the homogeneous pectin polysaccharide that the present embodiment obtains: esterification content is 63.61mg/g, and esterification degree is 35.0%; Acetylation content is 0mg/g, and acetylation degree is 0.

Embodiment 4: prepare homogeneous pectin polysaccharide TPS7-2A

41) will be dried tea leaf powder and be broken to 40～100 orders;

44) by step 43) efficient part that obtains dissolves by suitable quantity of water, centrifugal, gets supernatant liquor and is splined on sephadex chromatography post Superdex200, and water carries out wash-out; Utilize Shodex R1-102 differential refraction detector to detect and follow the tracks of the efficient part that elution curve (seeing shown in Fig. 4-1) is collected wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide TPS7-2A, total yield: 3.57wt%.

High performance liquid phase gel chromatography (HPGPC) figure that Fig. 4-2 are the homogeneous pectin polysaccharide TPS7-2A that obtains, can be illustrated by Fig. 4-2: the polysaccharide obtaining is homogeneous polysaccharide.

With reference to the molecular weight determination measuring and calculating described in embodiment 1, learn that the molecular-weight average of obtained homogeneous pectin polysaccharide TPS7-2A is 6.5 * 10 ⁴dalton.

With reference to the Structural Identification content described in embodiment 1, obtained homogeneous pectin polysaccharide TPS7-2A is carried out to Structural Identification:

After the complete acid hydrolysis of TPS7-2A, TLC analyzes and shows that it is acidic polysaccharose, containing uronic acid;

Fig. 4-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPS7-2A obtaining after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize; In b figure, be followed successively by from left to right rhamnosyl, Fucose, pectinose, wood sugar, seminose, glucose, semi-lactosi characteristic of correspondence peak; A figure is more known with b figure: in the GC collection of illustrative plates of the homogeneous pectin polysaccharide TPS7-2A obtaining after acetylize, to only have semi-lactosi characteristic peak, thereby can infer that TPS7-2A is comprised of galacturonic acid.

Fig. 4-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPS7-2A obtaining;

Fig. 4-5th, the GC-MS after the homogeneous pectin polysaccharide TPS7-2A obtaining methylates analyzes collection of illustrative plates, a figure is wherein the GC figure after homogeneous pectin polysaccharide TPS7-2A methylates, b figure is the corresponding mass spectrum in 26.53min peak in GC figure, and c figure is the corresponding mass spectrum in 31.64min peak in GC figure;

With reference to the galactouronic acid content assaying method described in embodiment 1, measuring and calculating learns that the galactouronic acid content in the homogeneous pectin polysaccharide that the present embodiment obtains is 87.6%.

With reference to the esterification described in embodiment 1 and acetylation analytical procedure, measure and to learn in the homogeneous pectin polysaccharide that the present embodiment obtains: esterification content is 17.19mg/g, and esterification degree is 9.5%; Acetylation content is 18.98mg/g, and acetylation degree is 57.2 ‰.

Embodiment 5: prepare homogeneous pectin polysaccharide TPSR4-1B

54) by step 53) efficient part that obtains dissolves by suitable quantity of water, centrifugal, gets supernatant liquor and is splined on propylene sephadex chromatography post Sephacryl S-300 High Resolution, and water carries out wash-out; Utilize Shodex R1-102 differential refraction detector to detect and follow the tracks of elution curve, according to elution curve (seeing shown in Fig. 5-1), collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide TPSR4-1B, total yield: 2.37wt%.

High performance liquid phase gel chromatography (HPGPC) figure that Fig. 5-2 are the homogeneous pectin polysaccharide TPSR4-1B that obtains, can be illustrated by Fig. 5-2: the polysaccharide obtaining is homogeneous polysaccharide.

With reference to the molecular weight determination measuring and calculating described in embodiment 1, learn that the molecular-weight average of obtained homogeneous pectin polysaccharide TPSR4-1B is 8.6 * 10 ⁵dalton.

With reference to the Structural Identification content described in embodiment 1, obtained homogeneous pectin polysaccharide TPSR4-1B is carried out to Structural Identification:

After the complete acid hydrolysis of TPSR4-1B, TLC analyzes and shows that it is acidic polysaccharose, containing uronic acid;

Fig. 5-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPSR4-1B obtaining after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize; In b figure, be followed successively by from left to right rhamnosyl, Fucose, pectinose, wood sugar, seminose, glucose, semi-lactosi characteristic of correspondence peak; A figure is more known with b figure: in the GC collection of illustrative plates of the homogeneous pectin polysaccharide TPSR4-1B obtaining after acetylize, to only have semi-lactosi characteristic peak, thereby can infer that TPSR4-1B is comprised of galacturonic acid.

Fig. 5-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPSR4-1B obtaining; From Fig. 5-4: in this polysaccharide, containing neutral sugar, (60-62ppm interval is not there are no CH ₂signal), forming result with monose coincide; 170ppm is galacturonic acid methyl esters carboxyl carbon signal, and anomeric carbon is positioned at 99-100ppm, shows that it is configured as α configuration, and 78ppm is 4 carbon signals of α-D galacturonic acid (and methyl esters), and 67-72ppm is C2, C3 and C5 signal, and 53ppm is methyl esters carbon signal.

Fig. 5-5th, the GC-MS after the homogeneous pectin polysaccharide TPSR4-1B obtaining methylates analyzes collection of illustrative plates, a figure is wherein the GC figure after homogeneous pectin polysaccharide TPSR4-1B methylates, b figure is the corresponding mass spectrum in 27.66min peak in GC figure, and c figure is the corresponding mass spectrum in 31.80min peak in GC figure; In sample from Fig. 5-5: TPSR4-1B after uronic acid reduction, only contain end group semi-lactosi (27.66min) and 1,4 semi-lactosis (31.8min) that connect, show that the sample of TPSR4-1B after uronic acid reduction is straight chain 1,4 Polygalactans that connect, thereby further show that TPSR4-1B is the polygalacturonic acid that straight chain Isosorbide-5-Nitrae connects.

Through periodate oxidation analysis, learn: in sample TPSR4-1B, the glycosyl of 1mol consumes the NaIO of 0.95mol ₄, show that its mode of connection is that Isosorbide-5-Nitrae connects.

To sum up structural analysis is learnt: the polygalacturonic acids homogeneous pectin polysaccharide that the polysaccharide that the present embodiment obtains also connects for Isosorbide-5-Nitrae.

With reference to the galactouronic acid content assaying method described in embodiment 1, measuring and calculating learns that the galactouronic acid content in the homogeneous pectin polysaccharide that the present embodiment obtains is 99.0%.

With reference to the esterification described in embodiment 1 and acetylation analytical procedure, measure and to learn in the homogeneous pectin polysaccharide that the present embodiment obtains: esterification content is 55.45mg/g, and esterification degree is 30.5%; Acetylation content is 4.22mg/g, and acetylation degree is 1.27 ‰.

Embodiment 6: prepare homogeneous pectin polysaccharide TPSR4-2A

64) by step 63) efficient part that obtains dissolves by suitable quantity of water, centrifugal, gets supernatant liquor and is splined on sephadex chromatography post Superdex200, and water carries out wash-out; Utilize Shodex R1-102 differential refraction detector to detect and follow the tracks of elution curve, according to elution curve (seeing shown in Fig. 6-1), collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide TPSR4-2A, total yield: 15.12wt%.

High performance liquid phase gel chromatography (HPGPC) figure that Fig. 6-2 are the homogeneous pectin polysaccharide TPSR4-2A that obtains, can be illustrated by Fig. 6-2: the polysaccharide obtaining is homogeneous polysaccharide.

With reference to the molecular weight determination measuring and calculating described in embodiment 1, learn that the molecular-weight average of obtained homogeneous pectin polysaccharide TPSR4-2A is 3.7 * 10 ⁵dalton.

With reference to the Structural Identification content described in embodiment 1, obtained homogeneous pectin polysaccharide TPSR4-2A is carried out to Structural Identification:

After the complete acid hydrolysis of TPSR4-2A, TLC analyzes and shows that it is acidic polysaccharose, containing uronic acid;

Fig. 6-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPSR4-2A obtaining after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize; In b figure, be followed successively by from left to right rhamnosyl, Fucose, pectinose, wood sugar, seminose, glucose, semi-lactosi characteristic of correspondence peak; A figure is more known with b figure: in the GC collection of illustrative plates of the homogeneous pectin polysaccharide TPSR4-2A obtaining after acetylize, to only have semi-lactosi characteristic peak, thereby can infer that TPSR4-2A is comprised of galacturonic acid.

Fig. 6-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPSR4-2A obtaining;

Fig. 6-5th, the GC-MS after the homogeneous pectin polysaccharide TPSR4-2A obtaining methylates analyzes collection of illustrative plates, a figure is wherein the GC figure after homogeneous pectin polysaccharide TPSR4-2A methylates, b figure is the corresponding mass spectrum in 27.68min peak in GC figure, and c figure is the corresponding mass spectrum in 31.90min peak in GC figure;

Through periodate oxidation analysis, learn: in sample TPSR4-2A, the glycosyl of 1mol consumes the NaIO of 0.98mol ₄, show that its mode of connection is that Isosorbide-5-Nitrae connects.

With reference to the galactouronic acid content assaying method described in embodiment 1, measuring and calculating learns that the galactouronic acid content in the homogeneous pectin polysaccharide that the present embodiment obtains is 98.5%.

With reference to the esterification described in embodiment 1 and acetylation analytical procedure, measure and to learn in the homogeneous pectin polysaccharide that the present embodiment obtains: esterification content is 51.51mg/g, and esterification degree is 28.3%; Acetylation content is 3.03mg/g, and acetylation degree is 9.15 ‰.

Embodiment 7: prepare homogeneous pectin polysaccharide TPSR7-1B

74) by step 73) efficient part that obtains dissolves by suitable quantity of water, centrifugal, gets supernatant liquor and is splined on propylene sephadex chromatography post Sephacryl S-300 High Resolution, and water carries out wash-out; Utilize Shodex R1-102 differential refraction detector to detect and follow the tracks of elution curve, according to elution curve (seeing shown in Fig. 7-1), collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide TPSR7-1B, total yield: 12.34wt%.

High performance liquid phase gel chromatography (HPGPC) figure that Fig. 7-2 are the homogeneous pectin polysaccharide TPSR7-1B that obtains, can be illustrated by Fig. 7-2: the polysaccharide obtaining is homogeneous polysaccharide.

With reference to the molecular weight determination measuring and calculating described in embodiment 1, learn that the molecular-weight average of obtained homogeneous pectin polysaccharide TPSR7-1B is 6.5 * 10 ⁴dalton.

With reference to the Structural Identification content described in embodiment 1, obtained homogeneous pectin polysaccharide TPSR7-1B is carried out to Structural Identification:

After the complete acid hydrolysis of TPSR7-1B, TLC analyzes and shows that it is acidic polysaccharose, containing uronic acid;

Fig. 7-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPSR7-1B obtaining after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize; In b figure, be followed successively by from left to right rhamnosyl, Fucose, pectinose, wood sugar, seminose, glucose, semi-lactosi characteristic of correspondence peak; A figure is more known with b figure: in the GC collection of illustrative plates of the homogeneous pectin polysaccharide TPSR7-1B obtaining after acetylize, to only have semi-lactosi characteristic peak, thereby can infer that TPSR7-1B is comprised of galacturonic acid.

Fig. 7-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPSR7-1B obtaining;

Fig. 7-5th, the GC-MS after the homogeneous pectin polysaccharide TPSR7-1B obtaining methylates analyzes collection of illustrative plates, a figure is wherein the GC figure after homogeneous pectin polysaccharide TPSR7-1B methylates, b figure is the corresponding mass spectrum in 27.80min peak in GC figure, and c figure is the corresponding mass spectrum in 32.01min peak in GC figure;

Through periodate oxidation analysis, learn: in sample TPSR7-1B, the glycosyl of 1mol consumes the NaIO of 1.03mol ₄, show that its mode of connection is that Isosorbide-5-Nitrae connects.

With reference to the galactouronic acid content assaying method described in embodiment 1, measuring and calculating learns that the galactouronic acid content in the homogeneous pectin polysaccharide that the present embodiment obtains is 100.0%.

With reference to the esterification described in embodiment 1 and acetylation analytical procedure, measure and to learn in the homogeneous pectin polysaccharide that the present embodiment obtains: esterification content is 60.56mg/g, and esterification degree is 33.3%; Acetylation content is 0mg/g, and acetylation degree is 0.

Embodiment 8: prepare homogeneous pectin polysaccharide TPSR7-2A

84) by step 83) efficient part that obtains dissolves with the sodium chloride aqueous solution of appropriate 0.2mol/L, centrifugal, gets supernatant liquor and is splined on sephadex chromatography post Superdex200, with the sodium chloride aqueous solution of 0.2mol/L, carries out wash-out; Utilize Shodex R1-102 differential refraction detector to detect and follow the tracks of elution curve, according to elution curve (seeing shown in Fig. 8-1), collect the efficient part of wash-out; Concentrated, lyophilize, obtains described homogeneous pectin polysaccharide TPSR7-2A, total yield: 15.89wt%.

High performance liquid phase gel chromatography (HPGPC) figure that Fig. 8-2 are the homogeneous pectin polysaccharide TPSR7-2A that obtains, can be illustrated by Fig. 8-2: the polysaccharide obtaining is homogeneous polysaccharide.

With reference to the molecular weight determination measuring and calculating described in embodiment 1, learn that the molecular-weight average of obtained homogeneous pectin polysaccharide TPSR7-2A is 2.8 * 10 ⁵dalton.

With reference to the Structural Identification content described in embodiment 1, obtained homogeneous pectin polysaccharide TPSR7-2A is carried out to Structural Identification:

After the complete acid hydrolysis of TPSR7-2A, TLC analyzes and shows that it is acidic polysaccharose, containing uronic acid;

Fig. 8-3rd, the GC collection of illustrative plates (a figure) of the homogeneous pectin polysaccharide TPSR7-2A obtaining after acetylize and GC collection of illustrative plates (b figure) comparison diagram of standard monose after acetylize; In b figure, be followed successively by from left to right rhamnosyl, Fucose, pectinose, wood sugar, seminose, glucose, semi-lactosi characteristic of correspondence peak; A figure is more known with b figure: in the GC collection of illustrative plates of the homogeneous pectin polysaccharide TPSR7-2A obtaining after acetylize, to only have semi-lactosi characteristic peak, thereby can infer that TPSR7-2A is comprised of galacturonic acid.

Fig. 8-4th, the carbon spectrogram of the homogeneous pectin polysaccharide TPSR7-2A obtaining;

Fig. 8-5th, the GC-MS after the homogeneous pectin polysaccharide TPSR7-2A obtaining methylates analyzes collection of illustrative plates, a figure is wherein the GC figure after homogeneous pectin polysaccharide TPSR7-2A methylates, b figure is the corresponding mass spectrum in 27.81min peak in GC figure, and c figure is the corresponding mass spectrum in 32.00min peak in GC figure;

Through periodate oxidation analysis, learn: in sample TPSR7-2A, the glycosyl of 1mol consumes the NaIO of 0.99mol ₄, show that its mode of connection is that Isosorbide-5-Nitrae connects.

With reference to the esterification described in embodiment 1 and acetylation analytical procedure, measure and to learn in the homogeneous pectin polysaccharide that the present embodiment obtains: esterification content is 47.40mg/g, and esterification degree is 26.1%; Acetylation content is 2.70mg/g, and acetylation degree is 7.97 ‰.

Finally be necessary described herein: above embodiment is only for being described in more detail technical scheme of the present invention; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.

Claims

1. a homogeneous pectin polysaccharide, is characterized in that: be that molecular-weight average is 2.7 * 10 ⁵dalton, galactouronic acid content is 90.7%, and esterification degree is 28.0%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 1.97 ‰ Isosorbide-5-Nitrae connects, and is designated as TPS4-1B; Its preparation method comprises the steps:

11) will be dried tea leaf powder and be broken to 40～100 orders;

2. a homogeneous pectin polysaccharide, is characterized in that: be that molecular-weight average is 3.1 * 10 ⁴dalton, galactouronic acid content is 100%, and esterification degree is 28.4%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 4.84 ‰ Isosorbide-5-Nitrae connects, and is designated as TPS4-2A; Its preparation method comprises the steps:

21) will be dried tea leaf powder and be broken to 40～100 orders;

3. a homogeneous pectin polysaccharide, is characterized in that: be that molecular-weight average is 1.0 * 10 ⁴dalton, galactouronic acid content is 99.5%, and esterification degree is 35.0%, and the polygalacturonic acids pectin polysaccharide that the Isosorbide-5-Nitrae that acetylation degree is 0 connects, is designated as TPS7-1B; Its preparation method comprises the steps:

31) will be dried tea leaf powder and be broken to 40～100 orders;

4. a homogeneous pectin polysaccharide, is characterized in that: be that molecular-weight average is 6.5 * 10 ⁴dalton, galactouronic acid content is 87.6%, and esterification degree is 9.5%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 57.2 ‰ Isosorbide-5-Nitrae connects, and is designated as TPS7-2A; Its preparation method comprises the steps:

41) will be dried tea leaf powder and be broken to 40～100 orders;

5. a homogeneous pectin polysaccharide, is characterized in that: be that molecular-weight average is 8.6 * 10 ⁵dalton, galactouronic acid content is 99.0%, and esterification degree is 30.5%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 1.27 ‰ Isosorbide-5-Nitrae connects, and is designated as TPSR4-1B; Its preparation method comprises the steps:

6. a homogeneous pectin polysaccharide, is characterized in that: be that molecular-weight average is 3.7 * 10 ⁵dalton, galactouronic acid content is 98.5%, and esterification degree is 28.3%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 9.15 ‰ Isosorbide-5-Nitrae connects, and is designated as TPSR4-2A; Its preparation method comprises the steps:

7. a homogeneous pectin polysaccharide, is characterized in that: be that molecular-weight average is 6.5 * 10 ⁴dalton, galactouronic acid content is 100%, and esterification degree is 33.3%, and the polygalacturonic acids pectin polysaccharide that the Isosorbide-5-Nitrae that acetylation degree is 0 connects, is designated as TPSR7-1B; Its preparation method comprises the steps:

8. a homogeneous pectin polysaccharide, is characterized in that: be that molecular-weight average is 2.8 * 10 ⁵dalton, galactouronic acid content is 100%, and esterification degree is 26.1%, and acetylation degree is the polygalacturonic acids pectin polysaccharide that 7.97 ‰ Isosorbide-5-Nitrae connects, and is designated as TPSR7-2A; Its preparation method comprises the steps: