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CN102827256A - Preparation and application of ginseng glycoprotein - Google Patents

Preparation and application of ginseng glycoprotein Download PDF

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Publication number
CN102827256A
CN102827256A CN2012103615757A CN201210361575A CN102827256A CN 102827256 A CN102827256 A CN 102827256A CN 2012103615757 A CN2012103615757 A CN 2012103615757A CN 201210361575 A CN201210361575 A CN 201210361575A CN 102827256 A CN102827256 A CN 102827256A
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genseng
molecular weight
ginseng
glycoprotein
acid
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姜瑞芝
王颖
陈英红
高其品
刘威
徐宏
罗浩铭
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姜瑞芝
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Abstract

The invention relates to the technical field of development of medicine and health food, and provides an ultrafiltration separation technique by a macroporous adsorbent resin and a hollow fiber column, particularly a method for extracting and separating ginseng glycoprotein from traditional Chinese medicine ginseng. In the glycoprotein, the total sugar content is 30% or so, the acidic sugar content is 10% or so, and the protein content is higher than 30%; the weight average molecular weight (Mw) of the ginseng glycoprotein is distributed at 1.7*10<4> or so; the glycosidic bond connecting mode of the saccharide part uses 1->4 connected glucose as the main chain; and the protein part is composed of 16 amino acids. The pharmacodynamical test provides that the ginseng glycoprotein has the anti-inflammatory, analgesic and depressant functions.

Description

Preparation of genseng gp and uses thereof
Technical field
The present invention relates to a kind of genseng gp that is obtained by extraction separation in the genseng, wherein total sugar content is about 30%, and acid sugar content is about 10%, and protein contnt is more than 30%, genseng gp weight-average molecular weight (M w) be distributed in 1.7 * 10 4About, the glycosidic link mode of connection of sugar moieties is a main chain for connecting glucose with 1 → 4, protein portion is made up of 16 seed amino acids.This genseng gp has anti-inflammatory, analgesia, sedative effect.The invention belongs to medicine and protective foods development field.
Background technology
Genseng is the dry root and rhizome of Araliaceae genseng (Panax ginseng C.A.Meyer).Be rare medicinal herbs, have unique pharmaceutical use and medical care effect, receive people's attention always deeply.Years of researches are the result show, genseng regulate cns, improve ability of learning and memory, improve heart function, aspects such as hypoglycemic activity, enhancing body's immunological function, antitumor action, antioxygenation all demonstrate very strong medical active.Up to now; Isolated chemical ingredients has saponins compound, polysaccharide (oligosaccharides) constituents, polypeptide, fatty acid compound, amino acid etc. from genseng, and wherein studying maximum is that ginsenoside class and polyose composition are the bioactive staple of genseng.At present, the research report to genseng gp is very few.Retrieve through document; Only find the separation and purification and the property research (pharmaceutical analysis magazine 2006 (26) 2 272-276 pages or leaves) thereof of the genseng gp of report such as Zhang Bin; The preparation of the genseng gp of document report is that genseng Crude polysaccharides extract is obtained first component peaks 1 through Hiprep2660Sephacryl S200 column chromatographic isolation and purification earlier, passes through HitrapQ again TM26/60 column chromatography gradient elution purifying obtains component P 1, detect this component P through HPGPC 1Be the symmetric protein-sugar component of a peak shape, weight-average molecular weight (M w) be distributed in about 24000, this component is dialysed to water, and lyophilize is genseng gp.The genseng gp of opening refined report only is one of them fragment of genseng gp (molecular weight is about 24000), and this segmental physico-chemical property is studied.And these claims 1 prepared genseng gp weight-average molecular weight (M w) be distributed in 1.7 * 10 4About, so these claims 1 have its essential difference with the genseng gp of opening refined report.
Resulting its weight-average molecular weight of genseng gp of the present invention (M w) be distributed in 1.7 * 10 4About, wherein the total sugar content in the genseng gp is about 30%, and acid sugar content is about 10%, and protein contnt is more than 30%.The resulting genseng gp of the present invention will be for being that development of raw materials new drug, protective foods or food have been widened development space with the genseng from now on.
Through the document retrieval, relevant gp biological activity report is a lot, mainly concentrates on activity research anti-oxidant, antitumor, the enhancing immunity aspect, and is very rare about the otherwise purposes of gp.Up to the present, do not retrieve the bioactive report of relevant genseng gp as yet.In the present invention, through the pharmacodynamic experiment digital proof genseng gp in anti-inflammatory, analgesia, activity aspect calm, wherein anti-inflammatory action is through confirming to the influence of mouse YLENE dropsy of ear with to the influence of mouse X 5189 foot swelling; Analgesic activity through Dichlorodiphenyl Acetate induced mice writhing response influence and the influence of Superlysoform induced mice pain confirmed; Sedative effect is through eclamptogenic influence confirms to the influence of mouse autonomic activities with to the mouse pentetrazole.Before the present invention, the research of the relevant genseng gp of Shang Weijian anti-inflammatory, analgesia, calm aspect report.Therefore, the content protected of claim of the present invention has novelty and novelty.
Summary of the invention
But one of the object of the invention has provided the genseng gp that the abundant industrialization of a kind of plant resources is produced, and development and utilization is celebrated and is the traditional Chinese medicine-genseng of " kings of hundred grass " better.This genseng gp is characterised in that: (a) total sugar content in the genseng gp is about 30%, and acid sugar content is about 10%, and protein contnt is more than 30%; (b) genseng gp weight-average molecular weight (M w) be distributed in 1.7 * 10 4About; (c) sugar moieties in the genseng gp is mainly glucose, in addition, also contains a spot of seminose, semi-lactosi, Fucose, galacturonic acid; (d) the glycosidic link mode of connection of sugar moieties is a main chain for connecting glucose with 1 → 4, and (e) protein portion is made up of 16 seed amino acids such as aspartic acid, L-glutamic acid, Serine, Histidine, glycocoll, l-arginine, Threonine, proline(Pro), L-Ala, Xie Ansuan, halfcystine, Isoleucine, leucine, phenylalanine(Phe), Methionin, network propylhomoserins.
Two of the object of the invention is the activity that filter out genseng gp through pharmacodynamics test; Show that through test-results genseng gp has good biological activity at anti-inflammatory, analgesia, aspect calm, lay a good foundation for genseng gp being developed to prophylactic treatment and the health care that new drug, protective foods or food is used for disease.
To achieve these goals, the invention provides following technical scheme:
1, the preparation of genseng gp
Genseng Panax ginseng C.A.Meyer is pulverized, add the decocting that 10-15 doubly measures and boil 2 times, each 3 hours, filter; Merging filtrate is concentrated into every 1ml and contains the 1g medicinal material, through the D101 macroporous adsorbent resin of having handled well; With 2 times of water elutions amount column volume, water elution liquid is that 20000 hollow fiber column carries out ultrafiltration through molecular weight cut-off, and it is that 1000 hollow fiber column carries out ultrafiltration that ultrafiltrated continues through molecular weight cut-off; The dense liquid that is equipped with concentrates, and vacuum-drying gets genseng gp.
2, the genseng glycoprotein structure characterizes
For the basic comprising of sugar in definite genseng gp with protein part, we utilize chemical process and spectroscopic data to verify that the product of above-mentioned explained hereafter is genseng gp, and analytical results is following:
(1) physico-chemical property result
The mensuration of total sugar content adopts the phenolsulfuric acid method, and glucose is as reference substance, the production standard curve, and the result shows that the total sugar content in the genseng gp is 34%.
Between the acid sugar Determination on content adopts-and the xenol method, galacturonic acid is as reference substance, the production standard curve, the result shows that the acid sugar content in the genseng gp is 12%,
The mensuration of protein contnt adopts the Lowry method, and bovine serum albumin is as reference substance, the production standard curve, and the result shows that the protein content in the genseng gp is 45%.
(2) MWD adopts HPLC, the differential detector, and the U.S. matches fragrant post SRT SEC-100 (molecular weight cut-off 100-100000), with tryptophane (molecular weight is 204.23), proteinase inhibitor (molecular weight is 6512), V B12(molecular weight is 1355), Lrax (molecular weight is 12327), bovine serum albumin (molecular weight is 66200) be as reference substance, the production standard curve, and the GPC computed in software is the result show, genseng gp weight-average molecular weight (M w) be distributed in 1.74 * 10 4About.
L n(M n)=Bo+B1×T+B2×T 2+B3×T 3
Bo=6.421077,?B1=-0.1460711,?B2=0,?B3=0
R 2=0.996563
(3) form glycan analysis and adopt the PMP-pre-column derivatization method; Sugar moieties in the genseng gp is prepared into verivate; With the verivate of each monose as reference substance; HPLC result shows that sugar moieties is mainly glucose in the genseng gp, also contains and a spot of seminose, semi-lactosi, Fucose and galacturonic acid composition.
(4) methylation analysis adopts the Hakomori method, and sugar moieties in the genseng gp is prepared into verivate, and GC-MS result shows that the glycosidic link mode of connection is a main chain for connecting glucose with 1 → 4 in the genseng gp.
(5) amino acid analysis; Adopt HPLC; With genseng gp with 6mol/L hydrochloric acid hydrolysis 20 hours, with 20 seed amino acids as reference substance, production standard curve respectively; The result shows that genseng gp is made up of aspartic acid, L-glutamic acid, Serine, Histidine, glycocoll, l-arginine, Threonine, proline(Pro), L-Ala, Xie Ansuan, halfcystine, Isoleucine, leucine, phenylalanine(Phe), Methionin, network propylhomoserin 16 seed amino acids.
Genseng gp has anti-inflammatory, analgesia, sedative effect, and these pharmacological actions are confirmed through following pharmacodynamics test example.
Oral and abdominal cavity gives mouse genseng gp, observes its influence to inflammation, pain and cns.Method: adopting mouse YLENE dropsy of ear and X 5189 foot swelling to lick metapedes as inflammatory model, mouse acetic acid twisting, formaldehyde is pain model, and mouse autonomic activities, pentetrazole cause to faint from fear makes calm model.
Animal: Kunming mouse, body weight 18-22g, male, available from Changchun Biological Products Institute, credit number: SCXK (Ji)-2006-0001.The drinking-water of freely ingesting.
Medicine: genseng gp is provided by Jilin Academy of Chinese Medicine Sciences plant chamber, is buff powder.Frosst) is a Shenyang Ao Jina pharmaceutcal corporation, Ltd product, lot number: 20120107.Antondin is He'nan Runhong Pharmaceutical Co., Ltd.'s product, lot number: 1206091.300,200,100mg/kg is genseng gp oral administration dosage, experiment oral administration previous day 2 times, each 8h at interval, experiment was administered once totally 3 times the same day again; 60,30,15mg/kg is genseng gp intraperitoneal administration dosage, only be administered once the same day in experiment.
The data statistics result handles with the t check.
Experimental technique and result
(1). anti-inflammatory action
A. to the influence of mouse YLENE dropsy of ear
Get 80 of male mices, be divided into oral group of control group, 50mg/kg Frosst), 300,200 at random, 100mg/kg is that oral group of genseng gp, 60,30,15mg/kg are genseng gp abdominal cavity group, 10 every group; Totally 8 groups; Behind oral administration 45min and intraperitoneal administration 30min, be coated with YLENE 20 μ l, put to death mouse behind the 20min, utilize 9mm iron to sweep away animal left and right sides ear with mouse left side ear; As the swelling degree, carry out " t " check with animal left and right sides ear difference.The result sees table 1.
B. to the influence of mouse X 5189 foot swelling
Grouping and administration are the same; The same day was injected 0.1% X 5189 0.03ml in every right back foot of mouse in experiment; 4h puts to death mouse behind the injection X 5189, before putting to death mouse 45,30min oral and intraperitoneal administration respectively, cuts left and right sides sole in sole in ankle fine hair boundary; As the swelling degree, carry out " t " check with the difference of left and right sides lumping weight amount.The result sees table 1,2.
Table 1. orally give genseng gp is to the influence of mouse inflammatory model
Figure 2012103615757100002DEST_PATH_IMAGE001
Annotate: compare with control group: * P 0.05, * * P 0.01, * * * P < 0.001.
Table 2. abdominal cavity gives the influence of genseng gp to the mouse inflammatory model
Figure 2012103615757100002DEST_PATH_IMAGE002
Annotate: compare with control group: * P 0.05, * * P 0.01, * * * P < 0.001.
From table 1,2 visible, mouse ear oedema and foot swelling due to genseng gp p-Xylol and the X 5189 all have certain restraining effect, explain that genseng gp has anti-inflammatory action.
(2) sedative effect
A. to the influence of mouse autonomic activities
Get 80 of mouse and mouse is divided into 8 groups at random by table 2; Every group 10; Positive drug intramuscular injection 0.15ml/kg antondin; Administration is the same, is placed in the autonomic activities registering instrument in oral administration 45min, abdominal cavity and intramuscular administration 30min, makes it adapt to the movable number of times in the 1min subsequently of opening entry behind 5 min.See table 2
Autonomic activities inhibiting rate (%)=(blank control group activity number of times-drug group activity number of times)/
Blank control group activity number of times * 100%.
B. to the eclamptogenic influence of mouse pentetrazole
The grouping administration is the same; Behind oral administration 40min, intramuscular injection and intraperitoneal administration 30min, each organizes ip pentetrazole 75 mg kg (75% faints from fear, and dosage takes place), and falling and present clonism with mouse is index; Observe and respectively organize convulsive attack number of times and latent period, see table 3.
Table 3. orally give genseng gp is to the sedative effect of mouse
Figure 2012103615757100002DEST_PATH_IMAGE003
Annotate: compare with control group: * P 0.05, * * P 0.01, * * * P < 0.001.
Table 4. abdominal cavity gives the sedative effect of genseng gp to mouse
Figure 2012103615757100002DEST_PATH_IMAGE004
Annotate: compare with control group: * P 0.05, * * P 0.01, * * * P < 0.001.
From table 3,4 visible, no matter oral still intraperitoneal administration genseng gp all can significantly reduce mouse autonomic activities number of times, and the convulsions that pentetrazole is caused also has the obvious suppression effect, proves that genseng gp has tangible sedative effect.
(3) pain effect
A. the influence of Dichlorodiphenyl Acetate induced mice writhing response
Get 80 of male mices, be divided into control group at random, 0.1ml/kg positive drug group.300,200,100mg/kg and 60,30,15mg/kg be oral and 6 the administration groups of intraperitoneal administration of genseng gp, totally 8 groups.Administration is the same, and behind oral administration 45min and intraperitoneal administration 30min, abdominal injection (ip) 0.9% acetic acid 0.2ml/ only writes down mouse writhing number of times in its 15min.The result sees table 3.
B. to the influence of Superlysoform induced mice pain
Get 80 of male mices, be divided into control group at random, 0.1ml/kg positive drug group, 300,200,100mg/kg and 60,30,15mg/kg be oral and 6 the administration groups of intraperitoneal administration of genseng gp, totally 8 groups.Administration is the same, behind oral administration 45min and intraperitoneal administration 30min, only injects 4% Superlysoform, 10 μ l/ down in the right back sufficient aponeurosis (aponeuroses) of mouse, writes down and reaches the number of times that the 10-20min mouse licks the right foot in back in its 2min.The result sees table 5,6.
Table 5. orally give genseng gp is to the analgesic activity of mouse
Figure 2012103615757100002DEST_PATH_IMAGE005
Annotate: compare with control group: * P 0.05, * * P 0.01, * * * P < 0.001.
Table 6. abdominal cavity gives the analgesic activity of genseng gp to mouse
Annotate: compare with control group: * P 0.05, * * P 0.01, * * * P < 0.001.
Table 5,6 shows that genseng gp is that the internal organs pain that Dichlorodiphenyl Acetate causes has significant analgesia role, also causes the pain due to the inflammation that significant analgesic activity is also arranged to proinflammatory agent Superlysoform, and it is suitable that it acts on antondin.
Can know that from above pharmacodynamic experiment genseng gp has significant effect in anti-inflammatory, analgesia, calmness.Be used to be developed to new drug or protective foods better curative effect will be arranged.
The present invention executes example through following experiment to be achieved.
Embodiment
Preparation technology's method is got genseng 5kg, is ground into meal, adds the water of 15 times of amounts; Decoct 2 times, each 3 hours, filter; Merging filtrate, filtrating is concentrated into 5 liters, through the D101 (internal diameter of having handled well: 10cm * height: 100cm) macroporous adsorbent resin; Water elution to 20 liter, water elution liquid are that 20000 hollow fiber column carries out ultrafiltration through molecular weight cut-off, and it is that 1000 hollow fiber column carries out ultrafiltration that ultrafiltrated continues through molecular weight cut-off; Ultrafiltrated concentrates, and vacuum-drying gets genseng gp 50 g.

Claims (3)

1. the preparation method of a genseng gp is characterized in that: genseng Panax ginseng C.A.Meyer is pulverized, add the decocting that 10-15 doubly measures and boil 2 times; Each 3 hours, filter merging filtrate; Be concentrated into every 1ml and contain the 1g medicinal material, through the D101 macroporous adsorbent resin of having handled well, with 2 times of amounts of water elution column volume; Water elution liquid is that 20000 hollow fiber column carries out ultrafiltration through molecular weight cut-off, and it is that 1000 hollow fiber column carries out ultrafiltration that ultrafiltrated continues through molecular weight cut-off, and the dense liquid that is equipped with concentrates; Vacuum-drying gets genseng gp.
2. according to the said genseng gp of claim 1, it is characterized in that:
(a) total sugar content in the genseng gp is about 30%, and acid sugar content is about 10%, and protein contnt is more than 30%;
(b) genseng gp weight-average molecular weight is distributed in 1.7 * 10 4About;
(c) sugar moieties in the genseng gp is mainly glucose, in addition, also contains a spot of seminose, semi-lactosi, Fucose, galacturonic acid;
(d) the glycosidic link mode of connection of sugar moieties is a main chain for connecting glucose with 1 → 4;
(e) protein part in the genseng gp is made up of aspartic acid, L-glutamic acid, Serine, Histidine, glycocoll, l-arginine, Threonine, proline(Pro), L-Ala, Xie Ansuan, halfcystine, Isoleucine, leucine, phenylalanine(Phe), Methionin, network propylhomoserin 16 seed amino acids.
3. according to the application of the said genseng gp of claim 2 in preparation anti-inflammatory, analgesia, sedative medicine and protective foods.
CN2012103615757A 2012-09-25 2012-09-25 Preparation and application of ginseng glycoprotein Pending CN102827256A (en)

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Cited By (8)

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Publication number Priority date Publication date Assignee Title
CN103435692A (en) * 2013-08-30 2013-12-11 姜瑞芝 Application of ginseng glycoprotein in medicaments and health-care foods for treating senile dementia
CN105175487A (en) * 2015-09-11 2015-12-23 北华大学 Ginseng protein with antioxidation activity and preparation method thereof
CN105833246A (en) * 2016-05-23 2016-08-10 吉林省中医药科学院 Application of enteric coated ginseng glycoprotein pellets to preparation of analgesic and sedative drug
CN107034257A (en) * 2016-11-23 2017-08-11 集安市清河人参交易市场有限公司 A kind of preparation technology of wild ginseng protein nano powder
CN111471090A (en) * 2020-04-23 2020-07-31 长春中医药大学 Ginseng glycoprotein and its preparation method and application
CN114577744A (en) * 2020-12-02 2022-06-03 山西振东制药股份有限公司 Method for determining total sugar content of compound sophora flavescens injection
WO2024040979A1 (en) * 2022-08-23 2024-02-29 沈阳药科大学 Ginseng acidic polysaccharide vaccine adjuvant, vaccine composition, and use thereof
WO2024066288A1 (en) * 2022-09-27 2024-04-04 沈阳药科大学 Vaccine adjuvant, vaccine composition, and use thereof

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103435692A (en) * 2013-08-30 2013-12-11 姜瑞芝 Application of ginseng glycoprotein in medicaments and health-care foods for treating senile dementia
CN103435692B (en) * 2013-08-30 2015-09-30 姜瑞芝 The application of ginseng candies albumen in the medicine and protective foods for the treatment of senile dementia
CN105175487A (en) * 2015-09-11 2015-12-23 北华大学 Ginseng protein with antioxidation activity and preparation method thereof
CN105833246A (en) * 2016-05-23 2016-08-10 吉林省中医药科学院 Application of enteric coated ginseng glycoprotein pellets to preparation of analgesic and sedative drug
CN107034257A (en) * 2016-11-23 2017-08-11 集安市清河人参交易市场有限公司 A kind of preparation technology of wild ginseng protein nano powder
CN111471090A (en) * 2020-04-23 2020-07-31 长春中医药大学 Ginseng glycoprotein and its preparation method and application
CN111471090B (en) * 2020-04-23 2022-02-08 长春中医药大学 Ginseng glycoprotein and preparation method and application thereof
CN114577744A (en) * 2020-12-02 2022-06-03 山西振东制药股份有限公司 Method for determining total sugar content of compound sophora flavescens injection
WO2024040979A1 (en) * 2022-08-23 2024-02-29 沈阳药科大学 Ginseng acidic polysaccharide vaccine adjuvant, vaccine composition, and use thereof
WO2024066288A1 (en) * 2022-09-27 2024-04-04 沈阳药科大学 Vaccine adjuvant, vaccine composition, and use thereof

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Application publication date: 20121219