CN110483657A - A kind of lobelia chinensis homogeneous polysaccharide and its preparation method and application - Google Patents
A kind of lobelia chinensis homogeneous polysaccharide and its preparation method and application Download PDFInfo
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- CN110483657A CN110483657A CN201910863145.7A CN201910863145A CN110483657A CN 110483657 A CN110483657 A CN 110483657A CN 201910863145 A CN201910863145 A CN 201910863145A CN 110483657 A CN110483657 A CN 110483657A
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- lobelia
- lobelia chinensis
- chinensis
- homogeneous
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- Granted
Links
- 240000003146 Lobelia chinensis Species 0.000 title claims abstract description 120
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- 238000002360 preparation method Methods 0.000 title claims abstract description 19
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 34
- 235000020824 obesity Nutrition 0.000 claims abstract description 34
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- 238000005349 anion exchange Methods 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 34
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 22
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 abstract description 14
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
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- 229930091371 Fructose Natural products 0.000 description 5
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 5
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- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
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- 241000196324 Embryophyta Species 0.000 description 2
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- YWEUIGNSBFLMFL-UHFFFAOYSA-N diphosphonate Chemical compound O=P(=O)OP(=O)=O YWEUIGNSBFLMFL-UHFFFAOYSA-N 0.000 description 2
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- DLYUQMMRRRQYAE-UHFFFAOYSA-N phosphorus pentoxide Inorganic materials O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 description 2
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Diabetes (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Polymers & Plastics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Obesity (AREA)
- Hematology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Materials Engineering (AREA)
- General Chemical & Material Sciences (AREA)
- Emergency Medicine (AREA)
- Child & Adolescent Psychology (AREA)
- Endocrinology (AREA)
- Sustainable Development (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Epidemiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The present invention provides a kind of lobelia chinensis homogeneous polysaccharides and its preparation method and application.Wherein, lobelia chinensis homogeneous polysaccharide preparation method the following steps are included: choose lobelia chinensis herb, be freeze-dried after carrying out water extract-alcohol precipitation to it, be made Chinese lobelia polysaccharide crude product；DEAE-Fast-Flow anion-exchange column is splined on after Chinese lobelia polysaccharide crude product is dissolved with water, it is eluted using distilled water, then it is eluted with the NaCl solution of various concentration, while being detected using phenol sulphate method, obtain lobelia chinensis water elution position polysaccharide；Lobelia chinensis water elution position polysaccharide is splined on sephadex chromatography column, is eluted using NaCl solution, active component is collected according to peak position out, is dialysed with bag filter, dialyzed solution freeze-drying obtains lobelia chinensis homogeneous polysaccharide.Lobelia chinensis homogeneous polysaccharide in the present invention can be substantially reduced the content of total cholesterol and triglycerides in obesity mice serum and liver, be used to prepare prevention or/and treat the medicament of obesity and hyperglycemia.
Description
Technical field
The present invention relates to lobelia chinensis homogeneous polysaccharide field more particularly to a kind of lobelia chinensis homogeneous polysaccharide and preparation method thereof and
Using.
Background technique
In recent years, obesity patient is more and more, and obese people is easier to cause hypertension, hyperlipidemia, hyperglycemia etc. " three
It is high " symptom, plant polyose, which has, adjusts blood glucose, and the biological activities such as reducing blood lipid can be used to improve and treat " three high " symptom.
Chinese medicine lobelia chinensis is Campanulaceae, has effects that clearing heat and detoxicating, inducing diuresis to remove edema, and modern science is to small molecule biology contained by it
Bases research is more, and especially plant polyose has become the research hotspot for finding the therapeutic agent of obesity.
Lobelia chinensis (Lobelia chinensis Lour) is Campanulaceae lobelia herbaceos perennial, also known as anxious solution
Rope, thin spartina and edible kernel of melon seeds grass.Originate in China East China and lower Yangtze and southern each province.Lobelia chinensis herb has clear for medication
The effect of thermal detoxification, inducing diuresis to remove edema and hemostasis.Early in just having in the written Compendium of Material Medica of Li Shizhen of the Ming Dynasty, " snake a poisonous snake mentioned in ancient books wound, smashes juice
Drink, it is applied with dregs " record.Lobelia chinensis smell is special, sweet in flavor and pungent, and Chinese medicine is made into medicine materical crude slice to be decocted in water for oral dose, by the dregs of a decoction with
Human milk is tuned into paste, spreads on affected part, and for treating carbuncle swells furunculosis, poisonous insect, poisonous snake are bitten, shingles zoster etc..
With the application of modern technologies, people have separated a variety of active ingredients in lobelia chinensis, mainly there is alkaloid
Class, flavonoids, saponins, amino acid, polysaccharide and some small molecule that can be used as the raw material of industry such as p-aminobenzoic acid prolong recklessly
Rope acid, succinic acid.Alkaloids and flavonoids are concentrated mainly on for the research of lobelia chinensis active constituent at present.For lobelia chinensis
The research of polysaccharide but not deeply, it is most for the research of Chinese lobelia polysaccharide all stop at polysaccharide extraction purification and technique it is excellent
Change, it is actually rare to the research of its bioactivity.It discloses in existing patent such as CN201110134569.3 from lobelia chinensis
The Chinese lobelia polysaccharide of middle extraction and its application disclose a kind of Chinese lobelia polysaccharide extracted from lobelia chinensis in CN102204946B
Application in preparation enhancing immune function drug.But so far, (1) has no that the structure to lobelia chinensis homogeneous polysaccharide is reported
Road；(2) research without lobelia chinensis homogeneous polysaccharide for bariatrician is reported；(3) without lobelia chinensis homogeneous polysaccharide for pre-
Anti- and treatment hyperglycemia research report.
Summary of the invention
The present invention is directed at least solve the problems, such as obesity and hyperglycemia existing in the prior art.The purpose of the present invention mentions
The compound of obesity and hyperglycemia can be effectively prevented, treated for one kind.
In a first aspect, the present invention provides a kind of lobelia chinensis homogeneous polysaccharide.
A kind of lobelia chinensis homogeneous polysaccharide, lobelia chinensis herb is after water extract-alcohol precipitation, column chromatographic isolation and purification obtains, and is linear straight
Chain polysaccharide, by end group alpha-D-glucose and with β -2, the D-Fructose residue of 1 glucosides key connection is formed, relative molecular weight 1-
20kDa.Preferably 2~4kDa, it is best with 2.7kD.
Second aspect, the present invention provide the preparation method of lobelia chinensis homogeneous polysaccharide.
The preparation method of lobelia chinensis homogeneous polysaccharide comprises the following specific steps that:
A) lobelia chinensis herb is taken, is freeze-dried after carrying out water extract-alcohol precipitation, Chinese lobelia polysaccharide crude product is made；
B) it is splined on anion-exchange column after being dissolved to step a) the Chinese lobelia polysaccharide crude product obtained with water, uses distilled water
It is eluted, is then eluted with NaCl solution, phend-sulphuric acid is detected, and lobelia chinensis water elution position polysaccharide is obtained；
C) step b) the lobelia chinensis water elution position polysaccharide obtained is splined on sephadex chromatography column, NaCl solution into
Row elution；Active component is collected according to peak position out, is dialysed with the bag filter that molecular cut off is 1000-14000Da, in dialysis
Liquid is freeze-dried to get the lobelia chinensis homogeneous polysaccharide.
In step a), ungrease treatment is carried out with ethyl alcohol to dry lobelia chinensis herb, carries out water extract-alcohol precipitation again after residue is dry.
The step a) includes following operation: the residue first to dry lobelia chinensis herb alcohol reflux degreasing, after degreasing
Water is added after drying to carry out heating extraction 2~5 times, combined extract is concentrated under reduced pressure, and supernatant is collected in centrifugation, is added 2~4
Amount ethyl alcohol carries out alcohol precipitation again, and the mass percent of ethyl alcohol is 90~100%, carries out vacuum freeze drying to the precipitating of collection, i.e.,
Obtain Chinese lobelia polysaccharide crude product.
The mass percent of ethyl alcohol used in ungrease treatment is 75%~100%.
Solid-liquid ratio (g/mL) when reflux degreasing is 1:5~1:20, is flowed back degreasing 1~20 hour.
The solid-liquid ratio (g/mL) of drying residue and the water being added every time after degreasing is 1:8~1:30, the temperature of heating extraction
Degree is 60~100 DEG C, and the time extracted every time is 1~5 hour.As preferred: the temperature of heating extraction is 85~100 DEG C, often
The time of secondary extraction is 1~3 hour.
When alcohol precipitation, the ethyl alcohol that the mass percent of 2~4 times of volumes is 90~100%, side edged are added into supernatant
Stirring stands 8~36 hours until the mass fraction of final ethyl alcohol is 75~85%.
The step b) includes following operation: anion friendship is splined on after Chinese lobelia polysaccharide crude product obtained is dissolved with water
Column is changed, is eluted using water, phend-sulphuric acid tracks elution curve, collects sugary soln, is concentrated under reduced pressure, concentrate dialysis
After be freeze-dried, obtain lobelia chinensis water elution position polysaccharide.
The anion-exchange column is DEAE-Fast Flow.
The step c) includes following operation: by lobelia chinensis water elution obtained position polysaccharide with 0.1~0.4mol/L's
NaCl solution dissolution, centrifugation take supernatant to be splined on sephadex chromatography column, with the NaCl solution of 0.1~0.4mol/L into
Row elution, is detected using differential refraction detector, collects active component according to peak position out；Concentration, uses molecular cut off
It dialyses, is freeze-dried to get the lobelia chinensis homogeneous polysaccharide for the bag filter of 1000-14000Da.
The third aspect, the present invention provide a kind of pharmaceutical composition.The pharmaceutical composition includes lobelia chinensis homogeneous polysaccharide.Root
According to the embodiment of the present invention, lobelia chinensis homogeneous polysaccharide can be substantially reduced total cholesterol and glycerol in obesity mice serum and liver
The content of three esters, and have no toxic side effect, pharmaceutical composition of the invention can effectively prevent, treat obesity；According to this hair
Bright embodiment, lobelia chinensis homogeneous polysaccharide can be substantially reduced the fasting blood-glucose of mouse, have extraordinary glucose tolerance, adjust
The effect of blood glucose is saved, pharmaceutical composition of the invention can effectively prevent, treat hyperglycemia.
Fourth aspect, the present invention provide the purposes in medicine preparation of lobelia chinensis homogeneous polysaccharide, and the drug is for pre-
Anti- and/or treatment obesity, the drug is for preventing and/or treating hyperglycemia.
5th aspect, the present invention provide a kind of food compositions.The food compositions include lobelia chinensis homogeneous polysaccharide.Root
According to the embodiment of the present invention, lobelia chinensis homogeneous polysaccharide can be substantially reduced total cholesterol and glycerol in obesity mice serum and liver
The content of three esters, and have no toxic side effect, food compositions of the invention effectively can prevent and/or improve obesity；According to
The embodiment of the present invention, lobelia chinensis homogeneous polysaccharide can be substantially reduced the fasting blood-glucose of mouse, have extraordinary glucose-tolerant
Property, the effect of blood glucose, the prevention hyperglycemia that food compositions of the invention can imitate are adjusted, and improve hyperglycemic symptoms.
6th aspect, present invention offer lobelia chinensis homogeneous polysaccharide are preparing the purposes in food, and the food is for pre-
Anti- and/or improvement obesity, the food is for preventing and/or improving hyperglycemic symptoms.
The dosage form of food compositions of the present invention or pharmaceutical composition can be it is diversified, as long as can make
Active constituent effectively reaches intracorporal dosage form and is all possible.For example it can be selected from: tablet, capsule, powder, granule, sugar
The common dosage forms such as slurry, solution, suspension, injection, tincture, oral solution, aerosol, mouth containing agent, electuary, pill, powder are received
The sustained-release dosage types such as metric system agent.
Compared with prior art, the present invention having the advantage that
(1) the lobelia chinensis homogeneous polysaccharide in the present invention can be substantially reduced total cholesterol in obesity mice serum and liver and
Triglycerides, and have no toxic side effect, it is expected to prevention is used to prepare as main or sole active agent or/and treats obesity
Health food or pharmaceutical preparation achieve conspicuousness progress and unexpected effect compared with the existing technology.
(2) lobelia chinensis homogeneous polysaccharide provided by the invention can be substantially reduced the fasting blood-glucose of mouse, have extraordinary Portugal
Grape sugared tolerance adjusts the effect of blood glucose, it is expected to be used to prepare prevention or/and the high blood for the treatment of as main or sole active agent
The health food or pharmaceutical preparation of sugar achieve conspicuousness progress and unexpected effect compared with the existing technology.
(3) preparation method of the present invention is simple, the lobelia chinensis homogeneous polysaccharide of available high-purity, is suitable for large-scale production.
Detailed description of the invention
Fig. 1 is the efficient liquid phase gel chromatography figure of the lobelia chinensis homogeneous polysaccharide obtained in the present invention；
Fig. 2 is the infared spectrum of the lobelia chinensis homogeneous polysaccharide obtained in the present invention；
Fig. 3 .1 is the hydrogen spectrum and carbon spectrogram spectrum of the lobelia chinensis homogeneous polysaccharide obtained in the present invention；
Fig. 3 .2 is the HSQC map of the lobelia chinensis homogeneous polysaccharide obtained in the present invention；
Fig. 3 .3 is the HHCOSY map of the lobelia chinensis homogeneous polysaccharide obtained in the present invention；
Fig. 4 .1 is the relational graph that the lobelia chinensis homogeneous polysaccharide obtained in the present invention influences TG (A) in obesity mice serum；
Fig. 4 .2 is the relational graph that the lobelia chinensis homogeneous polysaccharide obtained in the present invention influences TC (B) in obesity mice serum；
Fig. 5 .1 is the relational graph that the lobelia chinensis homogeneous polysaccharide obtained in the present invention influences TG (A) in obesity mice liver；
Fig. 5 .2 is the relational graph that the lobelia chinensis homogeneous polysaccharide obtained in the present invention influences TC (B) in obesity mice liver；
Fig. 6 is the relational graph that the lobelia chinensis homogeneous polysaccharide obtained in the present invention rings Mouse Liver ghost image；
Fig. 7 is the relationship that the lobelia chinensis homogeneous polysaccharide obtained in the present invention influences fat granule in obesity mice liver great Ye
Figure；
Fig. 8 is the pass that the lobelia chinensis homogeneous polysaccharide obtained in the present invention influences TG (A) in obesity mice serum and TC (B)
System's figure；
Fig. 9 is the measurement result figure of the lobelia chinensis homogeneous polysaccharide that obtains in the present invention to 5-8 weeks fasting blood-glucose of obesity mice.
Specific embodiment
For the benefit of to understanding of the invention, it is illustrated with reference to the accompanying drawings and embodiments.
A kind of preparation method of lobelia chinensis homogeneous polysaccharide is disclosed in the present invention, preparation method is simple, available height
The lobelia chinensis homogeneous polysaccharide of purity is suitable for large-scale production.
Embodiment one: the preparation method of lobelia chinensis homogeneous polysaccharide
(1) weigh dry lobelia chinensis herb 1kg, dissection, be added 10 times of amounts mass percent be 95~100% it is anhydrous
Alcohol reflux degreasing (remove low polar ingredient) flows back degreasing 2 hours at 80 DEG C, filters, obtains the residue insoluble in ethyl alcohol,
Dry residue, residue are added in Chinese medicine extracting tank in 50 DEG C of baking ovens, then 10 times of amount water, boil extraction twice, each 3h, mistake
Filter merges extracting solution twice, and the ethyl alcohol that mass percent is 95%~100% is added in concentration, stirring while adding, until final second
The mass percent of alcohol is diluted to 75%, stands alcohol precipitation 24 hours, and filtering collects and precipitates and fling to residual ethanol；To precipitate into
Row centrifugation, after centrifugation plus appropriate distilled water redissolves, and is freeze-dried in -80 DEG C, Chinese lobelia polysaccharide crude product 126g is made, yield is about
The 12.6% of herb quality；
(2) Chinese lobelia polysaccharide crude product 20g obtained is taken, 200mL distilled water is added to dissolve, is centrifuged (12000rpm, 10min),
Aspirate supernatant is splined on DEAE-Fast Flow column (10 × 40cm), is washed using distilled water and each concentration NaCl solution
It is de-, it is detected using sulfuric acid-phynol method, every pipe sugared content and in being detected 480nm in microplate reader, drafting elution curve, collection contains
Sugar juice is concentrated under reduced pressure, and is freeze-dried after the bag filter dialysis that concentrate is 3500Da with molecular cut off, lobelia chinensis water is made
Elute position polysaccharide；
(3) take lobelia chinensis water elution position polysaccharide 120mg obtained molten with the NaCl solution of 0.1,0.2 or 0.4mol/L
Solution, centrifugation, takes supernatant to be splined on sephadex chromatography column, is eluted with the NaCl solution of 0.1~0.4mol/L；It utilizes
Differential refraction detector detection collects active component according to peak position out；It is concentrated under reduced pressure, concentrate is with molecular cut off
Freeze-drying is after the bag filter dialysis of 3500Da to get the lobelia chinensis homogeneous polysaccharide (abbreviation LCPW).
To the identification and analysis of the lobelia chinensis homogeneous polysaccharide of preparation
(1) purity and molecular weight determination
Using gel chromatography (GPC) method: high performance liquid chromatograph (Shimadzu LC-10A)；Chromatographic column is KS805-804-802
It connects gel column (8.0 × 300mm)；Detector is Composition distribution；Mobile phase is the sodium-chloride water solution of 0.05mol/L；Column
Temperature: 40 DEG C；Flow velocity: 0.6mL/min；Sampling volume: 20 μ L.
Glucan known to molecular weight (Mw1152,5200,11600,23800,148000,273000,410000) is taken to make
For standard items, each 5mg is configured to the solution of 5mg/mL with sodium chloride (NaCl) aqueous solution of mobile phase 0.05mol/L respectively；With
10000rpm is centrifuged 10min；Take supernatant, each 20 μ L of sample introduction；Standard curve is done to retention time with the logarithm of molecular weight.
Sample to be tested 5mg is taken, the solution of 5mg/mL is configured to the sodium-chloride water solution of mobile phase 0.05mol/L；With
10000rpm is centrifuged 10min；Supernatant is taken, 20 μ L of sample introduction carries out the test of efficient liquid phase gel chromatography.
According to the sample retention time of measurement, the average molecular weight of sample can be acquired from standard curve.
Drawing modification and the molecular weight calculating of standard curve are automatically performed by GPC software.The lobelia chinensis that Fig. 1 is is uniform
Efficient liquid phase gel chromatography (HPGPC) figure of polysaccharide (LCPW), the single symmetrical peak on figure illustrate it for homogeneous polysaccharide.GPC is soft
The average molecular weight that part measures lobelia chinensis homogeneous polysaccharide (LCPW) is 2.7kDa.
(2) Structural Identification
1) sugared composition analysis:
Sugared composition analysis is carried out with Hydrolyze method: being taken polysaccharide sample 1mg, is dissolved in ampoule bottle with the TFA solution of 3ml, 2M, is sealed
Mouthful, 121 DEG C of hydrolysis 2h, taking-up, evaporated under reduced pressure, then plus methanol be evaporated three times with the TFA that goes out completely；Residue addition is gone after hydrolysis
Ionized water dissolution, crosses 0.22 μm of filter membrane.
Chromatographic condition is as follows:
Agilent highly effective liquid phase chromatographic system (Agilent 1100)；
Chromatographic column: NH2P-50 4E chromatographic column (250nm × 4.6nm)
Column temperature is 30 DEG C；Mobile phase is deionized water and acetonitrile (21:79；V/V) mixed solution, flow velocity 0.6mL/min,
Sample volume is 20 μ L；Detector uses ELSD evaporative light scattering detector.
Testing result: the Chinese lobelia polysaccharide as the result is shown go out two peaks, by with standard monosaccharide (including fructose)
Compare and Literature Consult, thus it is speculated that its may by fructose and glucose group at.
2) infrared spectrum analysis: taking sample 1mg to be dried in vacuum overnight in the drier for be equipped with phosphorus pentoxide, KBr pressure
Piece carries out infrared spectrum analysis.
The infared spectrum for the lobelia chinensis homogeneous polysaccharide (LCPW) that Fig. 2 is, in 3367cm-1There is an absorption intensity at place
Greatly, the wider characteristic absorption peak of peak value, it is clear that be due to caused by the hydroxyl stretching vibration of glycan molecule.In 2932cm-1Place has one
The weaker narrow peak of a absorption intensity, belongs to characteristic absorption peak caused by the stretching vibration of C-H on glycan molecule.The two absorption peak categories
In the characteristic absorption peak of compound of polysaccharide, and in 1639cm-1The absorption peak at place then belongs to the vibration of H-O-H in polysaccharide combination water
It is dynamic.1470cm-1~1200cm-1The less sharp signal peak of range is then as caused by the angle vibration of C-H.1200cm-1~
1000cm-1There are biggish signal peaks then to show the there is stretching vibration as caused by the C-O of C-O-H or C-O-C in range.In
936cm-1The absorption peak at place is the 874cm due to caused by the symmetrical stretching vibration of furan nucleus-1The absorption peak at place is the cross of-CH2
To caused by vibration, and 819cm-1Caused by the absorption peak at place is the C-H angle vibration of furan nucleus, these three characteristic peaks show
LCPW sample has the presence of furanose residue.
3) carbon spectrum analysis: taking sample 30mg to be placed in the drier for be placed with phosphorus pentoxide and be dried in vacuum overnight, and is added 400
μLD2O dissolution, surveys it at room temperature13C-NMR spectrum.
The hydrogen for the lobelia chinensis homogeneous polysaccharide LCPW that Fig. 3 .1 is is composed and carbon spectrogram spectrum, in Fig. 3 .1, LCPW's1H-NMR
Spectrogram shows that it has weaker signal peak in δ 5.44ppm, in conjunction with LCPW methylation analysis results it is found that δ 5.44ppm should
Be belong to α-D-Glcp-1 → anomer hydrogen.And there is stronger signal in δ 4.24,4.08 and δ, 3.98~3.68ppm range
Peak, independent basis1H-NMR is difficult to be belonged to, we analyze and belong to further in connection with its Fig. 3 .2 and 3.3 maps.
LCPW's13For C-NMR as shown in B in Fig. 3 .1, it is δ 104.5ppm respectively that in anomeric carbon signals area, there are two signal peaks
With δ 93.76ppm, their integrating peak areas ratio is about 14:1, so the signal peak in δ 104.5ppm belongs to the master of LCPW
Want anomeric carbon signals.According to the methylation analysis results of LCPW it is found that should belong to fructose residual in the signal peak of δ 104.5ppm
Base should belong to glucose residue in the signal peak of δ 93.76ppm.In addition, the different head signal of LCPW has occurred significantly downwards
Migration, shows that LCPW contains β type glycosidic bond.
For the DEPT-135 of LCPW as shown in C in Fig. 3 .1, the methylene signals peak at δ 62.13ppm should belong to fructose
The C1 of residue, the methylene signals peak at δ 63.34ppm should belong to the C6 of residue of fructose.
According to the HSQC (as shown in Figure 3 .2) of LCPW and1H-1H COSY (as shown in Fig. 3 .3) two dimension spectrogram, it is main
Residue1H NMR and13The chemical shift of C NMR belongs to respectively, as shown in table 1.
Its anomeric carbon area 1104.5 and 93.77ppm respectively correspond → 1)-Fruf- (2 → C-2 and Glcp- (1 → C-1
Signal.→ 1)-Fruf- (2 → in C-1, C-2, C-3, C-4, C-5 and C-6 signal respectively correspond 62.13,104.5,78.5,
75.5,82.3 and 63.34ppm.
According to above-mentioned analysis: it is that linear straight chain is more that the Chinese lobelia polysaccharide prepared in the embodiment of the present invention one, which is a kind of,
Sugar, by end group alpha-D-glucose and with β -2, it is homogeneous polysaccharide that the D-Fructose residue of 1 glucosides key connection, which forms,.
Table 1
Internal pharmacodynamic experiment of the lobelia chinensis homogeneous polysaccharide in obesity mouse in the present invention
(1) high lipid food feeds the foundation of mouse obesity disease model:
Animal: 6~8 week old, male ICR mouse 20；
Reagent: TG detection reagent and TC detection reagent；
Grouping: adaptable fed after a week, is randomly divided into 4 groups, every group of 5 mouse.First group (fatty with normal diet
10%, protein 20%, carbohydrate 70%) it feeds, excess-three group high lipid food (fat 60%, protein 20%, carbon
Hydrate 20%) it feeds, it does not limit and ingests and drink water, 25~30 DEG C of temperature, circadian rhythm rule is respectively 12h, cleans weekly
Mice rearing cage replaces a sawdust padding, records daily feed and averagely takes in, and surveys a weight weekly.Raising four
Zhou Hou, the mouse that one group of high lipid food of random selection is fed, with Chinese lobelia polysaccharide solution stomach-filling, dosage is 300mg/ (kgd),
It is denoted as BC group；The mouse for selecting one group of high lipid food to feed again, uses the stomach-filling of glimepiride tablet solution as positive control, dosage is
5mg/(kg·d)。
(2) measurement experiment mice serum TG and TC content
Blood collection is placed on 4 DEG C of refrigerators and stands in 1.5mL centrifuge tube, after the precipitation of blood cell sedimentation serum, uses TG
With the content of TC detection kit detection groups of animals Triglycerides in Serum and total cholesterol.
As a result: the testing result of TG and TC is as shown in Fig. 4 .1 and Fig. 4 .2 in groups of animals serum, compared with FC group, BC group
There is significant decrease with TG the and TC content of PC group, NC group is in normal level as Normal group, TG and TC content.
(3) liver TG and TC assay
After the completion of each group mouse is handled, takes the EP of 2mL to manage the liver 0.5g of interior mouse, 4.5mL dehydrated alcohol, In is added
Mechanical homogenisation under the conditions of ice-water bath, 2500rpm are centrifuged 10min, and supernatant TG and TC detection kit is taken to detect groups of animals
The content of triglycerides and total cholesterol in liver.
As a result: as shown in Fig. 5 .1 and Fig. 5 .2, the TG and TC of FC group contain the testing result of TG and TC in groups of animals liver
Measure more dispersed, this may be related with small white mouse itself adjusting, TG the and TC content balance FC group of BC group and PC group has significantly
It reduces, NC group is as Normal group, TG and TC content is in normal level, and dispersion degree is lower.
In conclusion LCPW has significant ground bioactivity for TG and TC content in obesity mice serum, this shows LCPW
Has the function of reducing blood lipid；And LCPW reduces the effect of TG and TC content in obesity mice liver, shows that LCPW can improve fertilizer
Fat disease patient organ is fat.
(4) liver weight and liver organization morphological feature
After anatomy experiment mouse takes out liver, it is put into physiological saline, washes away blood in liver, then suck life with filter paper
Salt water is managed, liver weight is weighed.Hepatomegaly leaf fixed in 10% formalin is taken out into production specimens paraffin embedding slices, after HE dyeing
With micro- sem observation, take pictures.
As a result: mouse liver weight as shown in fig. 6, the liver weight of FC group mouse is apparently higher than other three groups, BC group and
Relatively, and slightly above NC group, hereinbefore LCPW can improve obesity to the results show to the liver weight of PC group mouse
The correctness of this fat conclusion of disease patient organ.
It takes out liver after mouse dissection to visually observe, NC group mouse liver is bronzing, and edge is relatively thin and soft；FC
Group mouse liver is yellow-white, and edge is thicker and texture is greasy, whole looser；The liver of two groups of mouse of BC and PC is dark red
Color, edge is thicker, and surface is glossy.
The representative microscope photo of mouse liver great Ye is as shown in fig. 7, the HE stained slice of liver great Ye shows each group
The liver of mouse does not have inflammation, necrosis and Apoptosis.Wherein, the liver section of NC group mouse shows mouse liver framework in A
Completely, liver cell is normal；FC group mouse liver slice display mouse liver produces visible dispersivity cytoplasmic vacuoles in B
And steatosis, this imply that degeneration variation has occurred in liver, and there is a large amount of lipid drops in liver cell；BC in C
Group mouse liver slice display mouse liver shows moderate steatosis and there is lipid droplets in liver cell；PC in D
There is relatively small number of steatosises for group mouse liver slice display mouse liver, in its liver cell, there is a small amount of lipid
Drop exists.In summary experimental result, further demonstrating LCPW has weight-reducing, fat reducing and improves obesity patient's visceral obesity
Bioactivity.
Visible in summary: the novel synanthrin type lobelia chinensis that the present invention isolates and purifies out from Chinese medicine lobelia chinensis is uniform more
Sugar, the content of the total cholesterol and triglycerides that can be substantially reduced in obesity mice serum and liver, and have no toxic side effect, it is expected to
Prevention is used to prepare as main or sole active agent and/or treats the health food or pharmaceutical preparation of obesity.
Lobelia chinensis homogeneous polysaccharide is tested in the hypoglycemic drug effect of mouse in the present invention
(1) experimental material: small white mouse
(2) raising and modeling of mouse
20 adaptable feds of small white mouse after a week, are randomly divided into 4 groups, every group of 5 mouse.First group is used normal diet
(fat 10%, protein 20%, carbohydrate 70%) is fed, excess-three group high lipid food (fat 60%, protein
20%, carbohydrate 20%) it feeds, it does not limit and ingests and drink water, 25~30 DEG C of temperature, circadian rhythm rule, is respectively 12h,
A mice rearing cage is cleaned weekly, a sawdust padding is replaced, records daily feed and averagely take in, and surveys a body weekly
Weight.After raising four weeks, the mouse that one group of high lipid food of random selection is fed, with Chinese lobelia polysaccharide solution stomach-filling, dosage is
300mg/ (kgd), is denoted as BC group；The mouse for selecting one group of high lipid food to feed again uses the stomach-filling of glimepiride tablet solution as sun
Property control, dosage be 5mg/ (kgd), be denoted as PC group；It is remaining two groups, common to raise respectively with isometric physiological saline stomach-filling
Expect that nursing group as normal control, is denoted as NC group, high lipid food nursing group is denoted as FC group as obesity group.Gastric infusion is raised
It supports 5 weeks, surveys a mouse fasting blood-glucose (Fasting blood glucose, FBG) weekly within first 4 weeks, last week is taken orally
Glucose tolerance tests (Oral glucose tolerance test, OGTT) continue stomach-filling after OGTT experiment and give
Medicine is raised mouse one week, and experiment mice is then put to death, and mouse puts to death previous evening progress empty stomach processing.
(3) measurement of mouse fasting blood-glucose
Experiment mice carries out empty stomach processing in previous evening of taking a blood sample, and the next morning takes experiment mice tail vein blood one to drip,
It is measured with blood glucose meter.
As a result: mouse starts grouping administration on the 5th week after front is fed for 4 weeks with different feeds, and in the 5th, 6,7,8
Week measures the fasting blood-glucose of mouse respectively, and measurement result is as shown in A-D in Fig. 8, and the 5th week i.e. administration first week may be due to small
It is short that white mouse eats the high lipid food time, so the fasting blood-glucose content of two groups of administration group BC and PC is significantly lower than Normal group
NC, the normal fasting blood glucose level of mouse is in 3.89~6.11mol/L, FC and NC group is in the 5th week fasting blood-glucose all in this area
Between, illustrate that obesity models are established not yet；With the extension of feeding time, mouse obesity disease model is gradually formed, in Fig. 8
Shown in B and C, the fasting blood-glucose of FC group mouse is gradually risen, and is greater than 7mol/L in the 7th week fasting blood-glucose content, is exceeded threshold value, NC
The fasting blood-glucose of group maintains the normal level of 4~5mol/L always, and BC and two groups of PC of fasting blood-glucose content are more than NC group, but
Due to being administered, it is lower than FC group, and in normal level；At the 8th week, the fasting blood-glucose content of FC group mouse already close to
8mol/L, there were significant differences with it for other three groups blood-sugar contents, and within the scope of normal level fasting blood-glucose, this shows mouse
Obesity models are successfully established, and the blood-sugar content of BC group is less than 6.11mol/L, show that LCPW has the blood glucose of obesity mice
There is adjustment effect, the purpose for reducing its fasting blood-glucose can be played.
(4) Oral glucose tolerance test
Experiment mice be OGTT it is previous evening carry out empty stomach processing, the next morning, according to 2g/kg dosage give mouse use
Glucose solution stomach-filling takes each group mouse tail vein blood immediately later, with blood glucose meter measurement 0,30,60,90,120min
Blood-sugar content.
As a result: the Oral glucose tolerance experimental result of mouse is as shown in figure 9, give mouse glucose in stomach-filling
After 30min, there is peak value in blood-sugar content, and the blood-sugar content peak value highest of FC group, the blood-sugar content peak value of NC group is minimum, In
After 60min, the blood-sugar content of FC group still maintains higher level, other three groups of blood-sugar contents are begun to decline, and LCPW is herein
The increased effect of good inhibition blood glucose is shown, effect continues to comparable to antidiabetic drug Glimepiride, this inhibiting effect
120min makes blood glucose level be restored to initial measured value.In OGTT experiment, shown very with the BC group mouse of LCPW stomach-filling
Good glucose tolerance indicates that LCPW has and adjusts blood glucose, it is made to maintain the physiological action of normal level.
Visible in summary: the novel synanthrin type lobelia chinensis that the present invention isolates and purifies out from Chinese medicine lobelia chinensis is uniform more
Sugar can be substantially reduced the fasting blood-glucose of mouse, have extraordinary glucose tolerance, adjust the effect of blood glucose, it is expected to as
Main or sole active agent is used to prepare prevention and/or treats the health food or pharmaceutical preparation of hyperglycemia.
The dosage form of Chinese lobelia polysaccharide is prepared as active constituent in the present invention health food or pharmaceutical preparation can be more
Kind multiplicity, as long as active constituent can be made, which effectively to reach intracorporal dosage form, to be all possible.For example it can be selected from: tablet,
Capsule, powder, granule, syrup, solution, suspension, injection, tincture, oral solution, aerosol, mouth containing agent, electuary, ball
The sustained-release dosage types such as the common dosage forms such as agent, powder or nanometer formulation.
The above is only presently preferred embodiments of the present invention, not do limitation in any form to the present invention, although this
Invention has been disclosed in a preferred embodiment above, and however, it is not intended to limit the invention, any person skilled in the art, In
In the range of not departing from technical solution of the present invention, when the technology contents using the disclosure above make a little change or are modified to
With the equivalent embodiment of variation, but anything that does not depart from the technical scheme of the invention content, according to the technical essence of the invention to
Any simple modification, equivalent change and modification made by upper embodiment, all of which are still within the scope of the technical scheme of the invention.
It should be noted that this specification structure depicted in this specification institute accompanying drawings, ratio, size etc., only to cooperate
The bright revealed content of book is not intended to limit the invention enforceable limit so that those skilled in the art understands and reads
Fixed condition, therefore do not have technical essential meaning, the modification of any structure, the change of proportionate relationship or the adjustment of size, not
It influences still fall in disclosed technology contents under the effect of present invention can be generated and the purpose that can reach
In the range of capable of covering.
Claims (10)
1. a kind of lobelia chinensis homogeneous polysaccharide, it is characterised in that: lobelia chinensis herb is after water extract-alcohol precipitation, column chromatographic isolation and purification obtains
It arrives, is linear straight chain polysaccharide, by end group alpha-D-glucose and with β -2, the D-Fructose residue of 1 glucosides key connection is formed, and opposite point
Son amount is 1-20kDa.
2. a kind of method for preparing lobelia chinensis homogeneous polysaccharide described in claim 1, which is characterized in that the method includes as follows
Step:
A) lobelia chinensis herb is taken, is freeze-dried after carrying out water extract-alcohol precipitation, Chinese lobelia polysaccharide crude product is made；
B) it is splined on anion-exchange column after being dissolved to step a) the Chinese lobelia polysaccharide crude product obtained with water, is carried out using distilled water
Elution, is then eluted with NaCl solution, phend-sulphuric acid is detected, and lobelia chinensis water elution position polysaccharide is obtained；
C) step b) the lobelia chinensis water elution position polysaccharide obtained is splined on sephadex chromatography column, NaCl solution is washed
It is de-；Active component is collected according to peak position out, is dialysed with the bag filter that molecular cut off is 1000-14000Da, dialyzed solution is cold
It is lyophilized dry to get the lobelia chinensis homogeneous polysaccharide.
3. the method for preparation lobelia chinensis homogeneous polysaccharide according to claim 2, it is characterised in that: in step a), to drying
Lobelia chinensis herb carries out ungrease treatment with ethyl alcohol, carries out water extract-alcohol precipitation again after residue is dry.
4. the method for preparation lobelia chinensis homogeneous polysaccharide according to claim 2, it is characterised in that: the step a) includes such as
Lower operation: first to dry lobelia chinensis herb alcohol reflux degreasing, water, which is added, in the residue after degreasing after drying carries out heating and mentions
It taking 2~5 times, combined extract, is concentrated under reduced pressure, supernatant is collected in centrifugation, and 2~4 times of amount ethyl alcohol are added and carry out alcohol precipitation, ethyl alcohol
Mass percent is 90~100%, carries out vacuum freeze drying to the precipitating of collection to get Chinese lobelia polysaccharide crude product.
5. the method for preparation lobelia chinensis homogeneous polysaccharide according to claim 2, it is characterised in that: the step b) includes such as
Lower operation: it is splined on anion-exchange column after Chinese lobelia polysaccharide crude product obtained is dissolved with water, is eluted using water, benzene
Phenol-sulfuric acid method tracks elution curve, collects sugary soln, is concentrated under reduced pressure, and is freeze-dried after concentrate dialysis, obtains lobelia chinensis water
Elute position polysaccharide.
6. the method for preparation lobelia chinensis homogeneous polysaccharide according to claim 2, it is characterised in that: the step c) includes such as
Lower operation: the lobelia chinensis water elution obtained position polysaccharide NaCl solution of 0.1~0.4mol/L is dissolved, centrifugation takes supernatant
Liquid is splined on sephadex chromatography column, is eluted with the NaCl solution of 0.1~0.4mol/L, utilizes differential refraction detector
It is detected, active component is collected according to peak position out；Concentration, the bag filter for being 1000-14000Da with molecular cut off are saturating
Analysis is freeze-dried to get the lobelia chinensis homogeneous polysaccharide.
7. a kind of pharmaceutical composition, it is characterised in that: include lobelia chinensis homogeneous polysaccharide described in claim 1.
8. the purposes in medicine preparation of claim 1 lobelia chinensis homogeneous polysaccharide, it is characterised in that: the drug is for preventing
And/or treatment obesity, the drug is for preventing and/or treating hyperglycemia.
9. a kind of food compositions, it is characterised in that: include lobelia chinensis homogeneous polysaccharide described in claim 1.
10. claim 1 lobelia chinensis homogeneous polysaccharide is preparing the purposes in food, it is characterised in that: the food is for pre-
Anti- and/or improvement obesity, the food is for preventing and/or improving hyperglycemic symptoms.
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| CN114304631A (en) * | 2021-12-24 | 2022-04-12 | 湖南科尔生物技术有限公司 | Blood glucose reducing nutritional supplement and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970576A (en) * | 2005-06-08 | 2007-05-30 | 广东医学院 | Extraction and preparation process of polysaccharide of Pteris semipinnata L. with anticancer activity |
| CN102204946A (en) * | 2011-05-24 | 2011-10-05 | 河南中医学院 | Chinese lobelia polysaccharide extracted from Chinese lobelia and application thereof |
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2019
- 2019-09-12 CN CN201910863145.7A patent/CN110483657B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970576A (en) * | 2005-06-08 | 2007-05-30 | 广东医学院 | Extraction and preparation process of polysaccharide of Pteris semipinnata L. with anticancer activity |
| CN102204946A (en) * | 2011-05-24 | 2011-10-05 | 河南中医学院 | Chinese lobelia polysaccharide extracted from Chinese lobelia and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114304631A (en) * | 2021-12-24 | 2022-04-12 | 湖南科尔生物技术有限公司 | Blood glucose reducing nutritional supplement and preparation method and application thereof |
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